Animal protein-free pharmaceutical compositions

ABSTRACT

Animal protein-free, solid-form Clostridial toxin pharmaceutical compositions comprising a Clostridial toxin active ingredient and at least two excipients.

This application is a continuation of Ser. No. 17/071,239, filed Oct.15, 2020, which is a continuation of U.S. patent application Ser. No.16/925,667, filed Jul. 10, 2020, which is a continuation of U.S.application Ser. No. 13/427,582, filed Mar. 22, 2012, now abandoned,which is a continuation of U.S. patent application Ser. No. 12/331,816,filed Dec. 10, 2008, now U.S. Pat. No. 8,168,206, issued May 1, 2012,which is a continuation-in-part that claims priority pursuant to 35U.S.C. § 120 to U.S. patent application Ser. No. 11/524,683, filed Sep.21, 2006, now U.S. Pat. No. 8,137,677, issued Mar. 20, 2012, whichclaims priority pursuant to 35 U.S.C. § 119(e) to U.S. ProvisionalApplication No. 60/725,126, filed Oct. 6, 2005, each of which is herebyincorporated by reference in its entirety.

A pharmaceutical composition is a formulation comprises at least oneactive ingredient and at least one inert ingredient, called anexcipient, used as a diluent or vehicle for the active ingredient. Anexcipient is useful in one or more of the following as a stabilizingagent, a bulking agent, a cryo-protectant, a lyo-protectant, apreservative, and a buffer. A pharmaceutical composition can beprocessed into a solid form, such as, e.g., a lyophilized (freezedried), or vacuum dried powder which can be reconstituted with asuitable fluid, such as saline or water, prior to administration to apatient. Alternately, a pharmaceutical composition can be formulated asan aqueous solution or suspension.

The vast majority of pharmaceutical compositions include a smallmolecule (or chemical entity) as their active ingredient. Recently, withthe advent of the biotechnology industry, pharmaceutical compositionscomprising a protein active ingredient have been, or are currentlybeing, developed. Unfortunately, a protein active ingredient can be verydifficult to stabilize (i.e., maintained in a state where loss ofbiological activity is minimized), thereby resulting in a loss ofprotein and/or loss of protein activity during the formulation,reconstitution (if required) and storage of the pharmaceuticalcomposition prior to use. Stability problems can arise due to surfaceadsorption of a protein active ingredient, physical instability, suchas, e.g., denaturation or aggregation, or chemical instability, such as,e.g., cross-linking, deamidation, isomerization, oxidation, formation ofacidic or basic species, Maillard reaction, and fragmentation. Toprevent such instability, various protein-based excipients, such asalbumin and gelatin, have been used to stabilize a protein activeingredient present in a pharmaceutical composition.

Unfortunately, despite their known stabilizing effects, significantdrawbacks exist to the use of protein excipients, such as albumin orgelatin, in a pharmaceutical composition. For example albumin andgelatin are expensive and increasingly difficult to obtain. Furthermore,blood products or animal derived products such as albumin and gelatin,when administered to a patient can subject the patient to a potentialrisk of receiving blood borne pathogens or infectious agents. Thus, itis known that the possibility exists that the presence of ananimal-derived protein excipients in a pharmaceutical composition canresult in inadvertent incorporation of infectious elements into thepharmaceutical composition. For example, it has been reported that useof human serum albumin may transmit prions into a pharmaceuticalcomposition. Thus, it is desirable to find a suitable non-proteinexcipients, such as, e.g., stabilizers, cryo-protectants andlyo-protectants, which can be used to stabilize the protein activeingredient present in a pharmaceutical composition.

The unique characteristics of Clostridial toxins further constrain andhinder the selection of suitable non-protein excipients for apharmaceutical composition comprising a Clostridial toxin activeingredient. For example, Clostridial toxins are large proteins having anaverage molecular weight of approximately 150 kDa, and are furthercomplexed with non-toxin associated proteins that increase the size toapproximately 300-900-kDa. The size of a Clostridial toxin complex makesit much more fragile and labile than smaller, less complex proteins,thereby compounding the formulation and handling difficulties ifClostridial toxin stability is to be maintained. Hence, the use ofnon-protein excipients, such as, e.g., stabilizers, cryo-protectants andlyo-protectants must be able to interact with the Clostridial toxinactive ingredient in a manner which does not denature, fragment orotherwise inactivate the toxin or cause disassociation of the non-toxinassociated proteins present in the toxin complex.

Another problem associated with a Clostridial toxin active ingredient,is the exceptional safety, precision, and accuracy that is necessary forat all steps of the formulation process. Thus, a non-protein excipientshould not itself be toxic or difficult to handle so as to notexacerbate the already extremely stringent requirements currently inplace to formulate a pharmaceutical composition comprising a Clostridialtoxin active ingredient.

Still another difficulty linked with a Clostridial toxin activeingredient, is the incredible low amounts of Clostridial toxin that isused in a pharmaceutical composition. As with enzymes generally, thebiological activities of the Clostridial toxins are dependant, at leastin part, upon their three dimensional conformation. Thus, a Clostridialtoxin is detoxified by heat, various chemicals, surface stretching, andsurface drying. Additionally, it is known that dilution of a Clostridialtoxin complex obtained by the known culturing, fermentation andpurification methods to the much lower concentration used in apharmaceutical composition results in rapid inactivation of the toxin.The extremely low amount of a Clostridial toxin active ingredient thatis used in a pharmaceutical composition, makes this active ingredientvery susceptible to adsorption to, e.g., the surfaces of laboratoryglassware, vessels, to the vial in which the pharmaceutical compositionis reconstituted and to the inside surface of a syringe used to injectthe pharmaceutical composition. Such adsorption of a Clostridial toxinactive ingredient to surfaces can lead to a loss of active ingredientand to denaturation of the remaining Clostridial toxin activeingredient, both of which reduce the total activity of the activeingredient present in the pharmaceutical composition. Hence, the use ofnon-protein excipients, such as, e.g., stabilizers, cryo-protectants andlyo-protectants must be able to act as surface blockers to prevent theadsorption of a Clostridial toxin active ingredient to a surface. Todate, the only successful stabilizing agent for this purpose has beenthe animal derived proteins, such as, e.g., human serum albumin andgelatin.

Yet another problem connected to a Clostridial toxin active ingredient,is the pH-sensitivity associates with complex formation. For example,the 900-kDa BoNT/A complex is known to be soluble in dilute aqueoussolutions at pH 3.5-6.8. However, at a pH above about 7 the non-toxicassociated proteins dissociate from the 150-kDa neurotoxin, resulting ina loss of toxicity, particularly as the pH rises above pH 8.0. SeeEdward J. Schantz et al., pp. 44-45, Preparation and characterization ofbotulinum toxin type A for human treatment, in Jankovic, J., et al.,THERAPY WITH BOTULINUM TOXIN (Marcel Dekker, Inc., 1994). As thenon-toxic associated proteins are believed to preserve or help stabilizethe secondary and tertiary structures upon which toxicity is depends,the dissociation of these proteins results in a more unstableClostridial toxin active ingredient. Thus, non-protein excipients usefulto formulate a pharmaceutical composition comprising a Clostridial toxinactive ingredient must be able to operate within the confines of a pHlevel necessary to maintain the activity a Clostridial toxin activeingredient.

In light of the unique nature of Clostridial toxins and the requirementsset forth above, the probability of finding suitable non-proteinexcipients useful to formulate a pharmaceutical composition comprising aClostridial toxin active ingredient has been difficult. Prior to thepresent invention, only animal derived protein excipients, such as,e.g., human serum albumin and gelatin, were used successfully asstabilizers. Thus, albumin, by itself or with one or more additionalsubstances such as sodium phosphate or sodium citrate, is known topermit high recovery of toxicity of botulinum toxin type A afterlyophilization. Unfortunately, as already set forth, human serumalbumin, as a pooled blood product, can, at least potentially, carryinfectious or disease causing elements when present in a pharmaceuticalcomposition. Indeed, any animal product or protein such as human serumalbumin or gelatin can also potentially contain pyrogens or othersubstances that can cause adverse reactions upon injection into apatient.

What is needed therefore is a Clostridial toxin pharmaceuticalcompositions wherein the Clostridial toxin (such as a botulinum toxin)is stabilized by a non-protein excipient. The present invention relatesto Clostridial toxin pharmaceutical compositions with one or morenon-protein excipients which functions to stabilize the Clostridialtoxin present in the pharmaceutical composition.

Thus, in an aspect of the present invention, a Clostridial toxinpharmaceutical composition comprises an animal protein-free excipientand a Clostridial toxin active ingredient. In another aspect, aClostridial toxin pharmaceutical composition comprises at least two ananimal protein-free excipients and a Clostridial toxin activeingredient. In yet another aspect, a Clostridial toxin pharmaceuticalcomposition comprises at least three an animal protein-free excipientsand a Clostridial toxin active ingredient. A Clostridial toxin activeingredient can be a Clostridial toxin complex comprising theapproximately 150-kDa Clostridial toxin and other proteins collectivelycalled non-toxin associated proteins (NAPs), the approximately 150-kDaClostridial toxin alone, or a modified Clostridial toxin, such as, e.g.,a re-targeted Clostridial toxin.

Thus, in an aspect of the present invention, a Clostridial toxinpharmaceutical composition comprises a non-protein-based excipient and aClostridial toxin active ingredient. In another aspect, a Clostridialtoxin pharmaceutical composition comprises at least twonon-protein-based excipients and a Clostridial toxin active ingredient.In yet another aspect, a Clostridial toxin pharmaceutical compositioncomprises at least three non-protein-based excipients and a Clostridialtoxin active ingredient. A Clostridial toxin active ingredient can be aClostridial toxin complex comprising the approximately 150-kDaClostridial toxin and other proteins collectively called non-toxinassociated proteins (NAPs), the approximately 150-kDa Clostridial toxinalone, or a modified Clostridial toxin, such as, e.g., a re-targetedClostridial toxin.

In another aspect of the present invention, a Botulinum toxinpharmaceutical composition comprises an animal protein-free excipientand a Botulinum toxin active ingredient. In another aspect, a Botulinumtoxin pharmaceutical composition comprises at least two animalprotein-free excipients and a Botulinum toxin active ingredient. In yetanother aspect, a Botulinum toxin pharmaceutical composition comprisesat least three animal protein-free excipients and a Botulinum toxinactive ingredient. A Botulinum toxin active ingredient can be aBotulinum toxin complex comprising the approximately 150-kDa botulinumtoxin and NAPs, the 150-kDa Botulinum toxin alone, or a modifiedBotulinum toxin, such as, e.g., a re-targeted botulinum toxin.

In another aspect of the present invention, a Botulinum toxinpharmaceutical composition comprises a non-protein-based excipient and aBotulinum toxin active ingredient. In another aspect, a Botulinum toxinpharmaceutical composition comprises at least two non-protein-basedexcipients and a Botulinum toxin active ingredient. In yet anotheraspect, a Botulinum toxin pharmaceutical composition comprises at leastthree non-protein-based excipients and a Botulinum toxin activeingredient. A Botulinum toxin active ingredient can be a Botulinum toxincomplex comprising the approximately 150-kDa botulinum toxin and NAPs,the 150-kDa Botulinum toxin alone, or a modified Botulinum toxin, suchas, e.g., a re-targeted botulinum toxin.

Clostridia toxins produced by Clostridium botulinum, Clostridium tetani,Clostridium baratii and Clostridium butyricum are the most widely usedin therapeutic and cosmetic treatments of humans and other mammals.Strains of C. botulinum produce seven antigenically-distinct types ofBotulinum toxins (BoNTs), which have been identified by investigatingbotulism outbreaks in man (BoNT/A, /B, /E and/F), animals (BoNT/C,and/D), or isolated from soil (BoNT/G). BoNTs possess approximately 35%amino acid identity with each other and share the same functional domainorganization and overall structural architecture. It is recognized bythose of skill in the art that within each type of Clostridial toxinthere can be subtypes that differ somewhat in their amino acid sequence,and also in the nucleic acids encoding these proteins. For example,there are presently five BoNT/A subtypes, BoNT/A1, BoNT/A2, BoNT/A3,BoNT/A4 and BoNT/A5, with specific subtypes showing approximately 89%amino acid identity when compared to another BoNT/A subtype. While allseven BoNT serotypes have similar structure and pharmacologicalproperties, each also displays heterogeneous bacteriologicalcharacteristics. In contrast, tetanus toxin (TeNT) is produced by auniform group of C. tetani. Two other species of Clostridia, C. baratiiand C. butyricum, also produce toxins, BaNT and BuNT respectively, whichare similar to BoNT/F and BoNT/E, respectively.

Clostridial toxins are released by Clostridial bacterium as complexescomprising the approximately 150-kDa Clostridial toxin along withassociated non-toxin proteins (NAPs). Identified NAPs include proteinspossessing hemagglutination activity, such as, e.g., a hemagglutinin ofapproximately 17-kDa (HA-17), a hemagglutinin of approximately 33-kDa(HA-33) and a hemagglutinin of approximately 70-kDa (HA-70); as well asnon-toxic non-hemagglutinin (NTNH), a protein of approximately 130-kDa,see, e.g., Eric A. Johnson and Marite Bradshaw, Clostridial botulinumand its Neurotoxins: A Metabolic and Cellular Perspective, 39 Toxicon1703-1722 (2001); and Stephanie Raffestin et al., Organization andRegulation of the Neurotoxin Genes in Clostridium botulinum andClostridium tetani, 10 Anaerobe 93-100 (2004). Thus, the botulinum toxintype A complex can be produced by Clostridial bacterium as 900-kDa,500-kDa and 300-kDa forms. Botulinum toxin types B and C1 are apparentlyproduced as only a 500-kDa complex. Botulinum toxin type D is producedas both 300-kDa and 500-kDa complexes. Finally, botulinum toxin types Eand F are produced as only approximately 300-kDa complexes. Thedifferences in molecular weight for the complexes are due to differingratios of NAPs. The toxin complex is important for the intoxicationprocess because it provides protection from adverse environmentalconditions, resistance to protease digestion, and appears to facilitateinternalization and activation of the toxin.

Clostridial toxins are each translated as a single chain polypeptidethat is subsequently cleaved by proteolytic scission within a disulfideloop by a naturally-occurring protease. This cleavage occurs within thediscrete di-chain loop region created between two cysteine residues thatform a disulfide bridge. This posttranslational processing yields adi-chain molecule comprising an approximately 50 kDa light chain (LC)and an approximately 100 kDa heavy chain (HC) held together by thesingle disulfide bond and non-covalent interactions between the twochains. The naturally-occurring protease used to convert the singlechain molecule into the di-chain is currently not known. In someserotypes, such as, e.g., BoNT/A, the naturally-occurring protease isproduced endogenously by the bacteria serotype and cleavage occurswithin the cell before the toxin is release into the environment.However, in other serotypes, such as, e.g., BoNT/E, the bacterial strainappears not to produce an endogenous protease capable of converting thesingle chain form of the toxin into the di-chain form. In thesesituations, the toxin is released from the cell as a single-chain toxinwhich is subsequently converted into the di-chain form by anaturally-occurring protease found in the environment.

TABLE 1 Clostridial Toxin Reference Sequences and Regions H_(C) Toxin LCH_(N) H_(CN) H_(CC) BoNT/A M1-K448 A449-I873 I874-P1110 Y1111-L1296BoNT/B M1-K441 A442-I860 L861-E1097 Y1098-E1291 BoNT/C1 M1-K449T450-I868 N869-E1111 Y1112-E1291 BoNT/D M1-R445 D446-I864 N865-E1098Y1099-E1276 BoNT/E M1-R422 K423-I847 K848-E1085 Y1086-K1252 BoNT/FM1-K439 A440-I866 K867-K1105 Y1106-E1274 BoNT/G M1-K446 S447-I865S866-Q1105 Y1106-E1297 TeNT M1-A457 S458-L881 K882-E1127 Y1128-D1315BaNT M1-K431 N432-I857 I858-K1094 Y1095-E1268 BuNT M1-R422 K423-I847K848-E1085 Y1086-K1251

Each mature di-chain molecule comprises three functionally distinctdomains: 1) an enzymatic domain located in the LC that includes ametalloprotease region containing a zinc-dependent endopeptidaseactivity which specifically targets core components of theneurotransmitter release apparatus; 2) a translocation domain containedwithin the amino-terminal half of the HC (H_(N)) that facilitatesrelease of the LC from intracellular vesicles into the cytoplasm of thetarget cell; and 3) a binding domain found within the carboxyl-terminalhalf of the HC (H_(C)) that determines the binding activity and bindingspecificity of the toxin to the receptor complex located at the surfaceof the target cell. The H_(C) domain comprises two distinct structuralfeatures of roughly equal size that indicate function and are designatedthe H_(C)N and H_(Cc) subdomains. Table 1 gives approximate boundaryregions for each domain and subdomain found in exemplary Clostridialtoxins.

The binding, translocation and enzymatic activity of these threefunctional domains are all necessary for toxicity. While all details ofthis process are not yet precisely known, the overall cellularintoxication mechanism whereby Clostridial toxins enter a neuron andinhibit neurotransmitter release is similar, regardless of type.Although the applicants have no wish to be limited by the followingdescription, the intoxication mechanism can be described as comprisingat least four steps: 1) receptor binding, 2) complex internalization, 3)light chain translocation, and 4) enzymatic target modification. Theprocess is initiated when the H_(C) domain of a Clostridial toxin bindsto a toxin-specific receptor complex located on the plasma membranesurface of a target cell. The binding specificity of a receptor complexis thought to be achieved, in part, by specific combinations ofgangliosides and protein receptors that appear to distinctly compriseeach Clostridial toxin receptor complex. Once bound, the toxin/receptorcomplexes are internalized by endocytosis and the internalized vesiclesare sorted to specific intracellular routes. The translocation stepappears to be triggered by the acidification of the vesicle compartment.This process seems to initiate two important pH-dependent structuralrearrangements that increase hydrophobicity and promote formationdi-chain form of the toxin. Once activated, light chain endopeptidase ofthe toxin is released from the intracellular vesicle into the cytosolwhere it specifically targets one of three known core components of theneurotransmitter release apparatus. These core proteins,vesicle-associated membrane protein (VAMP)/synaptobrevin,synaptosomal-associated protein of 25 kDa (SNAP-25) and Syntaxin, arenecessary for synaptic vesicle docking and fusion at the nerve terminaland constitute members of the soluble N-ethylmaleimide-sensitivefactor-attachment protein-receptor (SNARE) family. BoNT/A and BoNT/Ecleave SNAP-25 in the carboxyl-terminal region, releasing a nine ortwenty-six amino acid segment, respectively, and BoNT/C1 also cleavesSNAP-25 near the carboxyl-terminus. BuNT cleaves at conserved portion ofSNAP-25 near the carboxyl-terminus. The botulinum serotypes BoNT/B,BoNT/D, BoNT/F and BoNT/G, TeNT, and BaNT act on the conserved centralportion of VAMP, and release the amino-terminal portion of VAMP into thecytosol. BoNT/C, cleaves syntaxin at a single site near the cytosolicmembrane surface. The selective proteolysis of synaptic SNAREs accountsfor the block of neurotransmitter release caused by Clostridial toxinsin vivo. The SNARE protein targets of Clostridial toxins are common toexocytosis in a variety of non-neuronal types; in these cells, as inneurons, light chain peptidase activity inhibits exocytosis, see, e.g.,Yann Humeau et al., How Botulinum and Tetanus Neurotoxins BlockNeurotransmitter Release, 82(5) Biochimie. 427-446 (2000); KathrynTurton et al., Botulinum and Tetanus Neurotoxins: Structure, Functionand Therapeutic Utility, 27(11) Trends Biochem. Sci. 552-558. (2002);Giovanna Lalli et al., The Journey of Tetanus and Botulinum Neurotoxinsin Neurons, 11(9) Trends Microbiol. 431-437, (2003).

The ability of Clostridial toxins, such as, e.g., BoNT/A, BoNT/B,BoNT/C₁, BoNT/D, BoNT/E, BoNT/F and BoNT/G, TeNT, BaNT and BuNT toinhibit neuronal transmission are being exploited in a wide variety oftherapeutic and cosmetic applications, see e.g., William J. Lipham,COSMETIC AND CLINICAL APPLICATIONS OF BOTULINUM TOXIN (Slack, Inc.,2004). Clostridial toxins commercially available as pharmaceuticalcompositions include, BoNT/A preparations, such as, e.g., BOTOX®(Allergan, Inc., Irvine, Calif.), Dysport®/Reloxin®, (Beaufour Ipsen,Porton Down, England), Neuronox® (Medy-Tox, Inc., Ochang-myeon, SouthKorea), BTX-A (Lanzhou Institute Biological Products, China) and Xeomin®(Merz Pharmaceuticals, GmbH., Frankfurt, Germany); and BoNT/Bpreparations, such as, e.g., MyoBloc™/NeuroBloc™ (SolsticeNeurosciences, Inc., South San Francisco, Calif.). As an example, BOTOX®is currently approved in one or more countries for the followingindications: achalasia, adult spasticity, anal fissure, back pain,blepharospasm, bruxism, cervical dystonia, essential tremor, glabellarlines or hyperkinetic facial lines, headache, hemifacial spasm,hyperactivity of bladder, hyperhidrosis, juvenile cerebral palsy,multiple sclerosis, myoclonic disorders, nasal labial lines, spasmodicdysphonia, strabismus and VII nerve disorder.

Aspects of the present pharmaceutical compositions provide, in part, aClostridial toxin pharmaceutical composition. As used herein, the term“Clostridial toxin pharmaceutical composition” refers to a formulationin which an active ingredient is a Clostridial toxin. As used herein,the term “formulation” means that there is at least one additionalingredient in the pharmaceutical composition besides a Clostridial toxinactive ingredient. A pharmaceutical composition is therefore aformulation which is suitable for diagnostic or therapeuticadministration to a subject, such as a human patient. The pharmaceuticalcomposition can be a solid formulation, such as, e.g., lyophilized(freeze-dried) or vacuum dried condition, or an aqueous formulation. Theconstituent ingredients of a pharmaceutical composition can be includedin a single composition (that is all the constituent ingredients, exceptfor any required reconstitution fluid, are present at the time ofinitial compounding of the pharmaceutical composition) or as atwo-component system, for example a vacuum-dried compositionreconstituted with a diluent such as saline which diluent contains aningredient not present in the initial compounding of the pharmaceuticalcomposition. A two-component system provides the benefit of allowingincorporation of ingredients which are not sufficiently compatible forlong-term shelf storage with the first component of the two componentsystem. For example, the reconstitution vehicle or diluent may include apreservative which provides sufficient protection against microbialgrowth for the use period, for example one-week of refrigerated storage,but is not present during the two-year freezer storage period duringwhich time it might degrade the toxin. Other ingredients, which may notbe compatible with a Clostridial toxin active ingredient or otheringredients for long periods of time, may be incorporated in thismanner; that is, added in a second vehicle (i.e. in the reconstitutionfluid) at the approximate time of use.

Aspects of the present pharmaceutical compositions provide, in part,animal protein-free. Clostridial toxin pharmaceutical composition. Asused herein, the term “animal protein-free” refers to the absence ofblood-derived, blood-pooled and other animal-derived products orcompounds. As used herein, the term “animal” refers to a mammal, bird,amphibian, reptile, fish, arthropod, or other animal species. “Animal”excludes plants and microorganisms, such as, e.g., yeast and bacteria.For example, an animal protein-free pharmaceutical composition can be apharmaceutical composition which is either substantially free oressentially free or entirely free of a serum derived albumin, gelatinand other animal-derived proteins, such as, e.g., immunoglobulins. Asused herein, the term “entirely free” (or “consisting of” terminology)means that within the detection range of the instrument or process beingused, the substance cannot be detected or its presence cannot beconfirmed. As used herein, the term “essentially free” (or “consistingessentially of”) means that only trace amounts of the substance can bedetected. As used herein, the term “substantially free” means present ata level of less than one percent by weight of the pharmaceuticalcomposition. As used herein, the term “animal-derived” refers to anycompounds or products purified directly from an animal source. As such,an animal protein recombinantly produced from a microorganism isexcluded from the term “animal-derived product or compound.” Thus,animal protein-free Clostridial toxin pharmaceutical compositions caninclude any of the Clostridial neurotoxin active ingredients disclosedin the present specification. As a non-limiting example of an animalprotein-free Clostridial toxin pharmaceutical composition is apharmaceutical composition comprising a BoNT/A toxin as the activeingredient and a suitable sugar and surfactant as excipients. As anothernon-limiting example of an animal protein-free Clostridial toxinpharmaceutical composition is a pharmaceutical composition comprising a900-kDa BoNT/A toxin complex as the active ingredient and a suitablesugar and surfactant as excipients. As yet another non-limiting exampleof an animal protein-free Clostridial toxin pharmaceutical compositionis a pharmaceutical composition comprising a modified BoNT/A toxinincluding an additional di-leucine motif as the active ingredient and asuitable sugar and surfactant as excipients. As still anothernon-limiting example of an animal protein-free Clostridial toxinpharmaceutical composition is a pharmaceutical composition comprising are-targeted BoNT/A including an opioid peptide targeting moiety as theactive ingredient and a suitable sugar and surfactant as excipients.

Aspects of the present pharmaceutical compositions provide, in part, aClostridial toxin active ingredient. As used herein, the term“Clostridial toxin active ingredient” refers to a therapeuticallyeffective concentration of a Clostridial toxin active ingredient, suchas, e.g., a Clostridial toxin complex, a Clostridial toxin, a modifiedClostridial toxin, or a re-targeted Clostridial toxin. As used herein,the term “therapeutically effective concentration” is synonymous with“therapeutically effective amount,” “effective amount,” “effectivedose,” “therapeutically effective dose” and refers to the minimum doseof a Clostridial toxin active ingredient necessary to achieve thedesired therapeutic effect and includes a dose sufficient to reduce asymptom associated with aliment being treated. In aspects of thisembodiment, a therapeutically effective concentration of a Clostridialtoxin active ingredient reduces a symptom associated with the alimentbeing treated by, e.g., at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90% or at least 100%. In other aspects of this embodiment, atherapeutically effective concentration of a Clostridial toxin activeingredient reduces a symptom associated with the aliment being treatedby, e.g., at most 10%, at most 20%, at most 30%, at most 40%, at most50%, at most 60%, at most 70%, at most 80%, at most 90% or at most 100%.

It is envisioned that any amount of Clostridial toxin active ingredientcan be added in formulating a Clostridial toxin pharmaceuticalcompositions disclosed in the present specification, with the provisothat a therapeutically effective amount of Clostridial toxin activeingredient is recoverable. In aspects of this embodiment, the amount ofClostridial toxin active ingredient added to the formulation is at least0.001 U/kg, at least 0.01 U/kg, at least 0.1 U/kg, at least 1.0 U/kg, atleast 10 U/kg, at least 100 U/kg, or at least 1000 U/kg. In otheraspects of this embodiment, the amount of Clostridial toxin activeingredient added to the formulation is at most 0.001 U/kg, at most 0.01U/kg, at most 0.1 U/kg, at most 1.0 U/kg, at most 10 U/kg, at most 100U/kg, or at most 1000 U/kg. In yet other aspects of this embodiment, theamount of Clostridial toxin active ingredient added to the formulationis from about 0.001 U/kg to about 1000 U/kg, about 0.01 U/kg to about1000 U/kg, about 0.1 U/kg to about 1000 U/kg, or about 1.0 U/kg to about1000 U/kg. In still other aspects of this embodiment, the amount ofClostridial toxin active ingredient added to the formulation is fromabout 0.001 U/kg to about 100 U/kg, about 0.01 U/kg to about 100 U/kg,about 0.1 U/kg to about 100 U/kg, or about 1.0 U/kg to about 100 U/kg.As used herein, the term “unit” or “U” is refers to the LD₅₀ dose, whichis defined as the amount of a Clostridial toxin, Clostridial toxincomplex or modified Clostridial toxin that killed 50% of the miceinjected with the Clostridial toxin, Clostridial toxin complex ormodified Clostridial toxin. As used herein, the term “about” whenqualifying a value of a stated item, number, percentage, or term refersto a range of plus or minus ten percent of the value of the stated item,percentage, parameter, or term.

In other aspects of this embodiment, the amount of Clostridial toxinactive ingredient added to the formulation is at least 1.0 pg, at least10 pg, at least 100 pg, at least 1.0 ng, at least 10 ng, at least 100ng, at least 1.0 μg, at least 10 μg, at least 100 pg, or at least 1.0mg. In still other aspects of this embodiment, the amount of Clostridialtoxin active ingredient added to the formulation is at most 1.0 pg, atmost 10 pg, at most 100 pg, at most 1.0 ng, at most 10 ng, at most 100ng, at most 1.0 μg, at most 10 μg, at most 100 μg, or at most 1.0 mg. Instill other aspects of this embodiment, the amount of Clostridial toxinactive ingredient added to the formulation is about 1.0 pg to about 10μg, about 10 pg to about 10 μg, about 100 pg to about 10 μg, about 1.0ng to about 10 μg, about 10 ng to about 10 μg, or about 100 ng to about10 μg. In still other aspects of this embodiment, the amount ofClostridial toxin active ingredient added to the formulation is about1.0 pg to about 1.0 μg, about 10 pg to about 1.0 μg, about 100 pg toabout 1.0 μg, about 1.0 ng to about 1.0 μg, about 10 ng to about 1.0 μg,or about 100 ng to about 1.0 μg.

Aspects of the present pharmaceutical compositions provide, in part, aClostridial toxin as a Clostridial toxin active ingredient. As usedherein, the term “Clostridial toxin” refers to any neurotoxin producedby a Clostridial toxin strain that can execute the overall cellularmechanism whereby a Clostridial toxin intoxicates a cell and encompassesthe binding of a Clostridial toxin to a low or high affinity Clostridialtoxin receptor, the internalization of the toxin/receptor complex, thetranslocation of the Clostridial toxin light chain into the cytoplasmand the enzymatic modification of a Clostridial toxin substrate.Non-limiting examples of Clostridial toxins include a Botulinum toxinlike BoNT/A, a BoNT/B, a BoNT/C₁, a BoNT/D, a BoNT/E, a BoNT/F, aBoNT/G, a Tetanus toxin (TeNT), a Baratii toxin (BaNT), and a Butyricumtoxin (BuNT). The BoNT/C2 cytotoxin and BoNT/C₃ cytotoxin, not beingneurotoxins, are excluded from the term “Clostridial toxin.” Clostridialtoxins can be obtained from, e.g., List Biological Laboratories, Inc.(Campbell, Calif.), the Centre for Applied Microbiology and Research(Porton Down, U.K), Wako (Osaka, Japan), and Sigma Chemicals (St Louis,Mo.). In addition, Clostridial toxins can be produced using standardpurification or recombinant biology techniques known to those skilled inthe art. For example, using the Schantz process, NAPs can be separatedout to obtain purified toxin, such as e.g., BoNT/A with an approximately150 kD molecular weight with a specific potency of 1-2×10⁸ LD₅₀ U/mg orgreater, purified BoNT/B with an approximately 156 kD molecular weightwith a specific potency of 1-2×10⁸ LD₅₀ U/mg or greater, and purifiedBoNT/F with an approximately 155 kD molecular weight with a specificpotency of 1-2×10⁷ LD₅₀ U/mg or greater. See Edward J. Schantz & Eric A.Johnson, Properties and use of Botulinum Toxin and Other MicrobialNeurotoxins in Medicine, Microbiol Rev. 56: 80-99 (1992). As anotherexample, recombinant Clostridial toxins can be recombinantly produced asdescribed in Lance E. Steward et al., Optimizing Expression of ActiveBotulinum Toxin Type A, U.S. Patent Publication 2008/0057575; and LanceE. Steward et al., Optimizing Expression of Active Botulinum Toxin TypeE, U.S. Patent Publication 2008/0138893, each of which is herebyincorporated in its entirety.

Any of a variety of sequence alignment methods can be used to determinepercent identity, including, without limitation, global methods, localmethods and hybrid methods, such as, e.g., segment approach methods.Protocols to determine percent identity are routine procedures withinthe scope of one skilled in the art and from the teaching herein.

Global methods align sequences from the beginning to the end of themolecule and determine the best alignment by adding up scores ofindividual residue pairs and by imposing gap penalties. Non-limitingmethods include, e.g., CLUSTAL W, see, e.g., Julie D. Thompson et al.,CLUSTAL W: Improving the Sensitivity of Progressive Multiple SequenceAlignment Through Sequence Weighting, Position-Specific Gap Penaltiesand Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680(1994); and iterative refinement, see, e.g., Osamu Gotoh, SignificantImprovement in Accuracy of Multiple Protein Sequence Alignments byIterative Refinement as Assessed by Reference to Structural Alignments,264(4) J. Mol. Biol. 823-838 (1996).

Local methods align sequences by identifying one or more conservedmotifs shared by all of the input sequences. Non-limiting methodsinclude, e.g., Match-box, see, e.g., Eric Depiereux and Ernest Feytmans,Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignmentof Several Protein Sequences, 8(5) CABIOS 501-509 (1992); Gibbssampling, see, e.g., C. E. Lawrence et al., Detecting Subtle SequenceSignals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131)Science 208-214 (1993); Align-M, see, e.g., Ivo Van Walle et al.,Align-M-A New Algorithm for Multiple Alignment of Highly DivergentSequences, 20(9) Bioinformatics: 1428-1435 (2004).

Hybrid methods combine functional aspects of both global and localalignment methods. Non-limiting methods include, e.g.,segment-to-segment comparison, see, e.g., Burkhard Morgenstern et al.,Multiple DNA and Protein Sequence Alignment Based On Segment-To-SegmentComparison, 93(22) Proc. Natl. Acad. Sci. U.S.A. 12098-12103 (1996);T-Coffee, see, e.g., Cédric Notredame et al., T-Coffee: A NovelAlgorithm for Multiple Sequence Alignment, 302(1) J. Mol. Biol. 205-217(2000); MUSCLE, see, e.g., Robert C. Edgar, MUSCLE: Multiple SequenceAlignment With High Score Accuracy and High Throughput, 32(5) NucleicAcids Res. 1792-1797 (2004); and DIALIGN-T, see, e.g., Amarendran RSubramanian et al., DIALIGN-T: An Improved Algorithm for Segment-BasedMultiple Sequence Alignment, 6(1) BMC Bioinformatics 66 (2005).

Thus in an embodiment, a Clostridial toxin pharmaceutical compositioncomprises a Clostridial toxin as the Clostridial toxin activeingredient. In aspects of this embodiment, a Clostridial toxinpharmaceutical composition comprises a BoNT/A, a BoNT/B, a BoNT/C1, aBoNT/D, a BoNT/E, a BoNT/F, a BoNT/G, a TeNT, a BaNT, or a BuNT. Inanother embodiment, a Clostridial toxin pharmaceutical compositioncomprises a Clostridial toxin variant as the Clostridial toxin activeingredient. In aspects of this embodiment, a Clostridial toxinpharmaceutical composition comprises naturally-occurring Clostridialtoxin variant or a non-naturally-occurring Clostridial toxin variant. Inother aspects of this embodiment, a Clostridial toxin pharmaceuticalcomposition comprises a BoNT/A variant, a BoNT/B variant, a BoNT/C1variant, a BoNT/D variant, a BoNT/E variant, a BoNT/F variant, a BoNT/Gvariant, a TeNT variant, a BaNT variant, or a BuNT variant, where thevariant is either a naturally-occurring variant or anon-naturally-occurring variant.

Aspects of the present pharmaceutical compositions provide, in part, aClostridial toxin complex as a Clostridial toxin active ingredient. Asused herein, the term “Clostridial toxin complex” refers to a complexcomprising a Clostridial toxin and associated NAPs, such as, e.g., aBotulinum toxin complex, a Tetanus toxin complex, a Baratii toxincomplex, and a Butyricum toxin complex. Non-limiting examples ofClostridial toxin complexes include those produced by a Clostridiumbotulinum, such as, e.g., a 900-kDa BoNT/A complex, a 500-kDa BoNT/Acomplex, a 300-kDa BoNT/A complex, a 500-kDa BoNT/B complex, a 500-kDaBoNT/C, complex, a 500-kDa BoNT/D complex, a 300-kDa BoNT/D complex, a300-kDa BoNT/E complex, and a 300-kDa BoNT/F complex. Clostridial toxincomplexes can be purified using the methods described in Schantz, supra,(1992); Hui Xiang et al., Animal Product Free System and Process forPurifying a Botulinum Toxin, U.S. Pat. No. 7,354,740, each of which ishereby incorporated by reference in its entirety. Clostridial toxincomplexes can be obtained from, e.g., List Biological Laboratories, Inc.(Campbell, Calif.), the Centre for Applied Microbiology and Research(Porton Down, U.K), Wako (Osaka, Japan), and Sigma Chemicals (St Louis,Mo.).

For example, high quality crystalline BoNT/A complex can be producedfrom the Hall A strain of Clostridium botulinum with characteristics of≥3×10⁷ U/mg, an A260/A278 of less than 0.60 and a distinct pattern ofbanding on gel electrophoresis using the Schantz process. See Schantz,supra, (1992). Generally, the BoNT/A complex can be isolated andpurified from an anaerobic fermentation by cultivating Clostridiumbotulinum type A in a suitable medium. Raw toxin can be harvested byprecipitation with sulfuric acid and concentrated byultramicrofiltration. Purification can be carried out by dissolving theacid precipitate in calcium chloride. The toxin can then be precipitatedwith cold ethanol. The precipitate can be dissolved in sodium phosphatebuffer and centrifuged. Upon drying there can then be obtainedapproximately 900 kD crystalline BoNT/A complex with a specific potencyof 3×10⁷ LD₅₀ U/mg or greater.

Thus in an embodiment, a Clostridial toxin pharmaceutical compositioncomprises a Clostridial toxin complex as the Clostridial toxin activeingredient. In aspects of this embodiment, a Clostridial toxinpharmaceutical composition comprises a BoNT/A complex, a BoNT/B complex,a BoNT/C, complex, a BoNT/D complex, a BoNT/E complex, a BoNT/F complex,a BoNT/G complex, a TeNT complex, a BaNT complex, or a BuNT complex. Inother aspects of this embodiment, a Clostridial toxin pharmaceuticalcomposition comprises a 900-kDa BoNT/A complex, a 500-kDa BoNT/Acomplex, a 300-kDa BoNT/A complex, a 500-kDa BoNT/B complex, a 500-kDaBoNT/C1 complex, a 500-kDa BoNT/D complex, a 300-kDa BoNT/D complex, a300-kDa BoNT/E complex, or a 300-kDa BoNT/F complex.

Aspects of the present pharmaceutical compositions provide, in part, amodified Clostridial toxin as a Clostridial toxin active ingredient. Asused herein, the term “modified Clostridial toxin” refers to anyClostridial toxin modified in some manner to provide a property orcharacteristic not present in the unmodified Clostridial toxin, but canstill execute the overall cellular mechanism whereby a Clostridial toxinintoxicates a cell, including, e.g., the binding of a Clostridial toxinto a low or high affinity Clostridial toxin receptor, theinternalization of the toxin/receptor complex, the translocation of theClostridial toxin light chain into the cytoplasm and the enzymaticmodification of a Clostridial toxin substrate. Non-limiting examples ofClostridial toxin variants are described in Steward, L. E. et al.,Post-Translational Modifications and Clostridial Neurotoxins, U.S. Pat.No. 7,223,577; Wei-Jen Lin et al., Neurotoxins with Enhanced TargetSpecificity, U.S. Pat. No. 7,273,722; Lance E. Steward et al.,Clostridial Neurotoxin Compositions and Modified Clostridial ToxinNeurotoxins, U.S. Patent Publication 2004/0220386; Steward, L. E. etal., Clostridial Toxin Activatable Clostridial Toxins, U.S. PatentPublication 2007/0166332; Lance E. Steward et al., Modified ClostridialToxins With Enhanced Targeting Capabilities For Endogenous ClostridialToxin Receptor Systems, U.S. Patent Publication 2008/0096248; Steward,L. E. et al., Modified Clostridial Toxins with Enhanced TranslocationCapability and Enhanced Targeting Activity, U.S. patent application Ser.No. 11/776,043; Steward, L. E. et al., Modified Clostridial Toxins withEnhanced Translocation Capabilities and Altered Targeting Activity ForClostridial Toxin Target Cells, U.S. patent application Ser. No.11/776,052; each of which is incorporated by reference in its entirety.Steward, L. E. et al., Degradable Clostridial Toxins, U.S. patentapplication Ser. No. 12/192,905; each of which is incorporated byreference in its entirety.

Thus in an embodiment, a Clostridial toxin pharmaceutical compositioncomprises a modified Clostridial toxin as the Clostridial toxin activeingredient. In aspects of this embodiment, a Clostridial toxinpharmaceutical composition comprises a modified BoNT/A, a modifiedBoNT/B, a modified BoNT/C₁, a modified BoNT/D, a modified BoNT/E, amodified BoNT/F, a modified BoNT/G, a modified TeNT, a modified BaNT, ora modified BuNT.

Aspects of the present pharmaceutical compositions provide, in part, are-targeted Clostridial toxin as a Clostridial toxin active ingredient.As used herein, the term “re-targeted Clostridial toxin” refers to aClostridial toxin modified to selectively bind to a non-Clostridialtoxin receptor present on a non-Clostridial toxin target cell, butotherwise execute the remaining intoxication steps of a Clostridialtoxin, such as, e.g., the internalization of the toxin/receptor complex,the translocation of the Clostridial toxin light chain into thecytoplasm and the enzymatic modification of a Clostridial toxinsubstrate. A retargeted Clostridial toxin can intoxicate wither aneuronal cell or a non-neuronal cell, depending on the modification madeto the Clostridial toxin. A re-targeted Clostridial toxin can be are-targeted Botulinum toxin, re-targeted Tetanus toxin, re-targetedBaratii toxin and a re-targeted Butyricum toxin. Non-limiting examplesof a re-targeted Clostridial toxin are described in, e.g., Keith A.Foster et al., Clostridial Toxin Derivatives Able To Modify PeripheralSensory Afferent Functions, U.S. Pat. No. 5,989,545; Clifford C. Shoneet al., Recombinant Toxin Fragments, U.S. Pat. No. 6,461,617; Conrad P.Quinn et al., Methods and Compounds for the Treatment of MucusHypersecretion, U.S. Pat. No. 6,632,440; Lance E. Steward et al.,Methods And Compositions For The Treatment Of Pancreatitis, U.S. Pat.No. 6,843,998; J. Oliver Dolly et al., Activatable RecombinantNeurotoxins, U.S. Pat. No. 7,132,259; Stephan Donovan, Clostridial ToxinDerivatives and Methods For Treating Pain, U.S. Patent Publication2002/0037833; Keith A. Foster et al., Inhibition of Secretion fromNon-neural Cells, U.S. Patent Publication 2003/0180289; Lance E. Stewardet al., Multivalent Clostridial Toxin Derivatives and Methods of TheirUse, U.S. Patent Publication 2006/0211619; Keith A. Foster et al.,Non-Cytotoxic Protein Conjugates, U.S. Patent Publication 2008/0187960;Steward, L. E. et al., Modified Clostridial Toxins with EnhancedTranslocation Capabilities and Altered Targeting Activity ForNon-Clostridial Toxin Target Cells, U.S. patent application Ser. No.11/776,075; Keith A. Foster et al., Re-targeted Toxin Conjugates, U.S.patent application Ser. No. 11/792,210; each of which is incorporated byreference in its entirety.

Thus in an embodiment, a Clostridial toxin pharmaceutical compositioncomprises a re-targeted Clostridial toxin as the Clostridial toxinactive ingredient. In aspects of this embodiment, a Clostridial toxinpharmaceutical composition comprises a re-targeted BoNT/A, a re-targetedBoNT/B, a re-targeted BoNT/C1, a re-targeted BoNT/D, a re-targetedBoNT/E, a re-targeted BoNT/F, a re-targeted BoNT/G, a re-targeted TeNT,a re-targeted BaNT, or a re-targeted BuNT. In another aspect of thisembodiment, a Clostridial toxin pharmaceutical composition comprises are-targeted Clostridial toxin comprises an opioid targeting moiety, suchas, e.g., an enkephalin, an endomorphin, an endorphin, a dynorphin, anociceptin or a hemorphin. In yet another aspect of this embodiment, aClostridial toxin pharmaceutical composition comprises a re-targetedClostridial toxin comprises a tachykinin targeting moiety, such as,e.g., a Substance P, a neuropeptide K (NPK), a neuropeptide gamma (NPgamma), a neurokinin A (NKA; Substance K, neurokinin alpha, neuromedinL), a neurokinin B (NKB), a hemokinin or a endokinin. In still anotheraspect of this embodiment, a Clostridial toxin pharmaceuticalcomposition comprises a re-targeted Clostridial toxin comprises amelanocortin targeting moiety, such as, e.g., a melanocyte stimulatinghormone, adrenocorticotropin, or a lipotropin. In still another aspectof this embodiment, a Clostridial toxin pharmaceutical compositioncomprises a re-targeted Clostridial toxin comprises a galanin targetingmoiety, such as, e.g., a galanin or a galanin message-associatedpeptide. In a further aspect of this embodiment, a Clostridial toxinpharmaceutical composition comprises a re-targeted Clostridial toxincomprises a granin targeting moiety, such as, e.g., a Chromogranin A, aChromogranin B, or a Chromogranin C. In another aspect of thisembodiment, a Clostridial toxin pharmaceutical composition comprises are-targeted Clostridial toxin comprises a Neuropeptide Y related peptidetargeting moiety, such as, e.g., a Neuropeptide Y, a Peptide YY,Pancreatic peptide ora Pancreatic icosapeptide. In yet another aspect ofthis embodiment, a Clostridial toxin pharmaceutical compositioncomprises a re-targeted Clostridial toxin comprises a neurohormonetargeting moiety, such as, e.g., a corticotropin-releasing hormone, aparathyroid hormone, a thyrotropin-releasing hormone, or a somatostatin.In still another aspect of this embodiment, a Clostridial toxinpharmaceutical composition comprises a re-targeted Clostridial toxincomprises a neuroregulatory cytokine targeting moiety, such as, e.g., aciliary neurotrophic factor, a glycophorin-A, a leukemia inhibitoryfactor, a cholinergic differentiation factor, an interleukin, anonostatin M, a cardiotrophin-1, a cardiotrophin-like cytokine, or aneuroleukin. In a further aspect of this embodiment, a Clostridial toxinpharmaceutical composition comprises a re-targeted Clostridial toxincomprises a kinin peptide targeting moiety, such as, e.g., a bradykinin,a kallidin, a desArg9 bradykinin, or a desArg10 bradykinin. In anotheraspect of this embodiment, a Clostridial toxin pharmaceuticalcomposition comprises a re-targeted Clostridial toxin comprises afibroblast growth factor targeting moiety, a nerve growth factortargeting moiety, an insulin growth factor targeting moiety, anepidermal growth factor targeting moiety, a vascular endothelial growthfactor targeting moiety, a brain derived neurotrophic factor targetingmoiety, a growth derived neurotrophic factor targeting moiety, aneurotrophin targeting moiety, such as, e.g., a neurotrophin-3, aneurotrophin-4/5, a head activator peptide targeting moiety, a neurturintargeting moiety, a persephrin targeting moiety, an artemin targetingmoiety, a transformation growth factor β targeting moiety, such as,e.g., a TGFβ1, a TGFβ2, a TGFβ3 or a TGFβ4, a bone morphogenic proteintargeting moiety, such as, e.g., a BMP2, a BMP3, a BMP4, a BMP5, a BMP6,a BMP7, a BMP8 or a BMP10, a growth differentiation factor targetingmoiety, such as, e.g., a GDF1, a GDF2, a GDF3, a GDF5, a GDF6, a GDF7, aGDF8, a GDF10, a GDF11 or a GDF15, or an activin targeting moiety, suchas, e.g., an activin A, an activin B, an activin C, an activin E or aninhibin A. In another aspect of this embodiment, a Clostridial toxinpharmaceutical composition comprises a re-targeted Clostridial toxincomprises a glucagon like hormone targeting moiety, such as, e.g., asecretin, a glucagon-like peptide, like a GLP-1 and a GLP-2, a pituitaryadenylate cyclase activating peptide targeting moiety, a growthhormone-releasing hormone targeting moiety, vasoactive intestinalpeptide targeting moiety like a VIP1 or a VIP2, a gastric inhibitorypolypeptide targeting moiety, a calcitonin-related peptidesvisceral gutpeptide targeting moiety like a gastrin, a gastrin-releasing peptide ora cholecystokinin, or a PAR peptide targeting moiety like a PAR1peptide, a PAR2 peptide, a PAR3 peptide or a PAR4 peptide.

Aspects of the present pharmaceutical compositions provide, in part, apharmacologically acceptable excipient. As used herein, the term“pharmacologically acceptable excipient” is synonymous with“pharmacological excipient” or “excipient” and refers to any excipientthat has substantially no long term or permanent detrimental effect whenadministered to mammal and encompasses compounds such as, e.g.,stabilizing agent, a bulking agent, a cryo-protectant, a lyo-protectant,an additive, a vehicle, a carrier, a diluent, or an auxiliary. Anexcipient generally is mixed with an active ingredient, or permitted todilute or enclose the active ingredient and can be a solid, semi-solid,or liquid agent. It is also envisioned that a pharmaceutical compositioncomprising a Clostridial toxin active ingredient can include one or morepharmaceutically acceptable excipients that facilitate processing of anactive ingredient into pharmaceutically acceptable compositions. Insofaras any pharmacologically acceptable excipient is not incompatible withthe Clostridial toxin active ingredient, its use in pharmaceuticallyacceptable compositions is contemplated. Non-limiting examples ofpharmacologically acceptable excipients can be found in, e.g.,Pharmaceutical Dosage Forms and Drug Delivery Systems (Howard C. Anselet al., eds., Lippincott Williams & Wilkins Publishers, 7^(th) ed.1999); Remington: The Science and Practice of Pharmacy (Alfonso R.Gennaro ed., Lippincott, Williams & Wilkins, 20th ed. 2000); Goodman &Gilman's The Pharmacological Basis of Therapeutics (Joel G. Hardman etal., eds., McGraw-Hill Professional, 10^(th) ed. 2001); and Handbook ofPharmaceutical Excipients (Raymond C. Rowe et al., APhA Publications,4th edition 2003), each of which is hereby incorporated by reference inits entirety.

Aspects of the present pharmaceutical compositions provide, in part, aneffective amount.” As used herein, the term “effective amount,” whenused in reference to the amount of an excipient or specific combinationof excipients added to a Clostridial toxin composition, refers to theamount of each excipient that is necessary to achieve the desiredinitial recovered potency of a Clostridial toxin active ingredient. Inaspects of this embodiment, an effective amount of an excipient orcombination of excipients results in an initial recovered potency of,e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90% or at least100%. In other aspects of this embodiment, a therapeutically effectiveconcentration of a Clostridial toxin active ingredient reduces a symptomassociated with the aliment being treated by, e.g., at most 10%, at most20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, atmost 80%, at most 90% or at most 100%.

In yet aspects of this embodiment, an effective amount of an excipientis, e.g., at least 0.1 mg, at least 0.125 mg, at least 0.2 mg, at least0.25 mg, at least 0.3 mg, at least 0.3125 mg, at least 0.4 mg, at least0.5 mg, at least 0.6 mg, at least 0.625 mg, at least 0.7 mg, at least0.8 mg, or at least 0.9 mg. In still aspects of this embodiment, aneffective amount of an excipient is, e.g., at least 1.0 mg, at least 2.0mg, at least 3.0 mg, at least 4.0 mg, at least 5.0 mg, at least 6.0 mg,at least 7.0 mg, at least 8.0 mg, or at least 9.0 mg. In further aspectsof this embodiment, an effective amount of an excipient is, e.g., atleast 10 mg, at least 20 mg, at least 30 mg, at least 40 mg, at least 50mg, at least 60 mg, at least 70 mg, at least 80 mg, at least 90 mg, orat least 100 mg.

In yet aspects of this embodiment, an effective amount of an excipientis, e.g., at most 0.1 mg, at most 0.125 mg, at most 0.2 mg, at most 0.25mg, at most 0.3 mg, at most 0.3125 mg, at most 0.4 mg, at most 0.5 mg,at most 0.6 mg, at most 0.625 mg, at most 0.7 mg, at most 0.8 mg, or atmost 0.9 mg. In still aspects of this embodiment, an effective amount ofan excipient is, e.g., at most 1.0 mg, at most 2.0 mg, at most 3.0 mg,at most 4.0 mg, at most 5.0 mg, at most 6.0 mg, at most 7.0 mg, at most8.0 mg, or at most 9.0 mg. In further aspects of this embodiment, aneffective amount of an excipient is, e.g., at most 10 mg, at most 20 mg,at most 30 mg, at most 40 mg, at most 50 mg, at most 60 mg, at most 70mg, at most 80 mg, at most 90 mg, or at most 100 mg.

In yet aspects of this embodiment, an effective amount of an excipientis, e.g., from about 0.1 mg to about 100 mg, from about 0.1 mg to about50 mg, from about 0.1 mg to about 10 mg, from about 0.25 mg to about 100mg, from about 0.25 mg to about 50 mg, from about 0.25 mg to about 10mg, from about 0.5 mg to about 100 mg, from about 0.5 mg to about 50 mg,from about 0.5 mg to about 10 mg, from about 0.75 mg to about 100 mg,from about 0.75 mg to about 50 mg, from about 0.75 mg to about 10 mg,from about 1.0 mg to about 100 mg, from about 1.0 mg to about 50 mg, orfrom about 1.0 mg to about 10 mg.

Aspects of the present pharmaceutical compositions provide, in part,non-protein excipient. As used herein, the term “non-protein excipient”refers to any excipient that is not a polypeptide comprising at leastfifteen amino acids. It is envisioned that any non-protein excipient isuseful in formulating a Clostridial toxin pharmaceutical compositionsdisclosed in the present specification, with the proviso that atherapeutically effective amount of the Clostridial toxin activeingredient is recovered using this non-protein excipient.

Aspects of the present pharmaceutical compositions provide, in part, asugar. As used herein, the term “sugar” refers to a compound comprisingone to 10 monosaccharide units, e.g., a monosaccharide, a disaccharide,a trisaccharide, and an oligosaccharide comprising four to tenmonosaccharide units. It is envisioned that any sugar is useful informulating a Clostridial toxin pharmaceutical compositions disclosed inthe present specification, with the proviso that a therapeuticallyeffective amount of the Clostridial toxin active ingredient is recoveredusing this sugar. Monosaccharides are polyhydroxy aldehydes orpolyhydroxy ketones with three or more carbon atoms, including aldoses,dialdoses, aldoketoses, ketoses and diketoses, as well as cyclic forms,deoxy sugars and amino sugars, and their derivatives, provided that theparent monosaccharide has a (potential) carbonyl group. Monosaccharidesinclude trioses, like glyceraldehyde and dihydroxyacetone; tetroses,like erythrose, threose and erythrulose; pentoses, like arabinose,lyxose, ribose, xylose, ribulose, xylulose; hexoses, like allose,altrose, galactose, glucose, gulose, idose, mannose, talose, fructose,psicose, sorbose, tagatose, fucose, rhamnose; heptoses, likesedoheptulose and mannoheptulose; octooses, like octulose and2-keto-3-deoxy-manno-octonate; nonoses like sialose; and decose.Oligosaccharides are compounds in which at least two monosaccharideunits are joined by glycosidic linkages. According to the number ofunits, they are called disaccharides, trisaccharides, tetrasaccharides,pentasaccharides, hexosaccharides, heptosaccharides, octosaccharides,nonosaccharides, decosaccharides, etc. An oligosaccharide can beunbranched, branched or cyclic. Common disaccharides include, withoutlimitation, sucrose, lactose, maltose, trehalose, cellobiose,gentiobiose, kojibiose, laminaribiose, mannobiose, melibiose, nigerose,rutinose, and xylobiose. Common trisaccharides include, withoutlimitation, raffinose, acarbose, maltotriose, and melezitose. Othernon-limiting examples of specific uses of sugar excipients can be foundin, e.g., ANSEL, SUPRA, (1999); GENNARO, SUPRA, (2000); HARDMAN, SUPRA,(2001); AND ROWE, SUPRA, (2003), each of which is hereby incorporated byreference in its entirety.

Thus in an embodiment, a Clostridial toxin pharmaceutical compositioncomprises a sugar. In aspects of this embodiment, a Clostridial toxinpharmaceutical composition comprises a monosaccharide. In other aspectsof this embodiment, a Clostridial toxin pharmaceutical compositioncomprises a disaccharide, a trisaccharide, a tetrasaccharide, apentasaccharide, a hexosaccharide, a heptosaccharide, an octosaccharide,a nonosaccharide, or a decosaccharide. In yet other aspects of thisembodiment, a Clostridial toxin pharmaceutical composition comprises anoligosaccharide comprising two to ten monosaccharide units.

It is envisioned that any amount of sugar is useful in formulating aClostridial toxin pharmaceutical compositions disclosed in the presentspecification, with the proviso that a therapeutically effective amountof the Clostridial toxin active ingredient is recovered using this sugaramount. In aspects of this embodiment, the amount of sugar added to theformulation is at least 0.5% (w/v), at least 1.0% (w/v), at least 2.0%(w/v), at least 3.0% (w/v), at least 4.0% (w/v), at least 5.0% (w/v), atleast 6.0% (w/v), at least 7.0% (w/v), at least 8.0% (w/v), at least9.0% (w/v), at least 10% (w/v), at least 15% (w/v), at least 20% (w/v),at least 25% (w/v), at least 30% (w/v), or at least 35% (w/v). In otheraspects of this embodiment, the amount of sugar added to the formulationis at most 0.5% (w/v), at most 1.0% (w/v), at most 2.0% (w/v), at most3.0% (w/v), at most 4.0% (w/v), at most 5.0% (w/v), at most 6.0% (w/v),at most 7.0% (w/v), at most 8.0% (w/v), at most 9.0% (w/v), at most 10%(w/v), at most 15% (w/v), at most 20% (w/v), at most 25% (w/v), at most30% (w/v), or at most 35% (w/v).

Aspects of the present pharmaceutical compositions provide, in part, apolyol. As used herein, the term “polyol” is synonymous with “sugaralcohol,” “polyhydric alcohol,” and “polyalcohol” and refers to a sugarderivative having an alcohol group (CH₂OH) instead of the aldehyde group(CHO), such as, e.g., mannitol from mannose, xylitol from xylose, andlactitol from lactulose. It is envisioned that any polyol is useful informulating a Clostridial toxin pharmaceutical compositions disclosed inthe present specification, with the proviso that a therapeuticallyeffective amount of the Clostridial toxin active ingredient is recoveredusing this polyol. Non-limiting examples of polyols include, glycol,glycerol, arabitol, erythritol, xylitol, maltitol, sorbitol (glucitol),mannitol, inositol, lactitol, galactitol (iditol), isomalt. Othernon-limiting examples of sugar excipients can be found in, e.g., Ansel,supra, (1999); Gennaro, supra, (2000); Hardman, supra, (2001); and Rowe,supra, (2003), each of which is hereby incorporated by reference in itsentirety.

Thus in an embodiment, a Clostridial toxin pharmaceutical compositioncomprises a polyol. In aspects of this embodiment, a Clostridial toxinpharmaceutical composition comprises glycol, glycerol, arabitol,erythritol, xylitol, maltitol, sorbitol (glucitol), mannitol, inositol,lactitol, galactitol (iditol), or isomalt.

It is envisioned that any amount of polyol is useful in formulating aClostridial toxin pharmaceutical compositions disclosed in the presentspecification, with the proviso that a therapeutically effective amountof the Clostridial toxin active ingredient is recovered using thispolyol amount. In aspects of this embodiment, the amount of polyol addedto the formulation is at least 0.5% (w/v), at least 1.0% (w/v), at least2.0% (w/v), at least 3.0% (w/v), at least 4.0% (w/v), at least 5.0%(w/v), at least 6.0% (w/v), at least 7.0% (w/v), at least 8.0% (w/v), atleast 9.0% (w/v), at least 10% (w/v), at least 15% (w/v), at least 20%(w/v), at least 25% (w/v), at least 30% (w/v), or at least 35% (w/v). Inother aspects of this embodiment, the amount of polyol added to theformulation is at most 0.5% (w/v), at most 1.0% (w/v), at most 2.0%(w/v), at most 3.0% (w/v), at most 4.0% (w/v), at most 5.0% (w/v), atmost 6.0% (w/v), at most 7.0% (w/v), at most 8.0% (w/v), at most 9.0%(w/v), at most 10% (w/v), at most 15% (w/v), at most 20% (w/v), at most25% (w/v), at most 30% (w/v), or at most 35% (w/v).

Aspects of the present pharmaceutical compositions provide, in part, apolymer. As used herein, the term “polymer” refers to high molecularweight compounds comprising at least eleven monomeric units. Polymersconsisting of only one kind of repeating unit are called homopolymers,whereas polymers formed from two or more different repeating units andcalled copolymers. A polymer can be natural or synthetic. Non-limitingexamples of polymers include polysaccharides, such as, e.g., dextrans(like dextran 1K, dextran 4K, dextran 40K, dextran 60K, and dextran70K), dextrin, glycogen, inulin, starch, starch derivatives (likehydroxymethyl starch, hydroxyethyl starch, hydroxypropyl starch,hydroxybutyl starch, and hydroxypentyl starch), hetastarch, cellulose,FICOLL, methyl cellulose (MC), carboxymethyl cellulose (CMC),hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC),hydroxyethyl methyl cellulose (HEMC), hydroxypropyl methyl cellulose(HPMC); polyvinyl acetates (PVA); polyvinyl pyrrolidones (PVP), alsoknown as povidones, having a K-value of less than or equal to 18, aK-value greater than 18 or less than or equal to 95, or a K-valuegreater than 95, like PVP 12 (KOLLIDON® 12), PVP 17 (KOLLIDON® 17), PVP25 (KOLLIDON® 25), PVP 30 (KOLLIDON® 30), PVP 90 (KOLLIDON® 90);polyethylene glycols like PEG 100, PEG 200, PEG 300, PEG 400, PEG 500,PEG 600, PEG 700, PEG 800, PEG 900, PEG 1000, PEG 1100, PEG 1200, PEG1300, PEG 1400, PEG 1500, PEG 1600, PEG 1700, PEG 1800, PEG 1900, PEG2000, PEG 2100, PEG 2200, PEG 2300, PEG 2400, PEG 2500, PEG 2600, PEG2700, PEG 2800, PEG 2900, PEG 3000, PEG 3250, PEG 3350, PEG 3500, PEG3750, PEG 4000, PEG 4250, PEG 4500, PEG 4750, PEG 5000, PEG 5500, PEG6000, PEG 6500, PEG 75000, PEG 7500, or PEG 8000; and polyethyleneimines (PEI); polypeptides (proteins) like bovine serum albumin,gelatin, and ovalbumin; polynucleotides like DNA and RNA. Othernon-limiting examples of polymer excipients can be found in, e.g.,Ansel, supra, (1999); Gennaro, supra, (2000); Hardman, supra, (2001);and Rowe, supra, (2003), each of which is hereby incorporated byreference in its entirety.

It is envisioned that any non-protein polymer is useful in formulating aClostridial toxin pharmaceutical compositions disclosed in the presentspecification, with the proviso that a therapeutically effective amountof the Clostridial toxin active ingredient is recovered using thisnon-protein polymer. Thus in an embodiment, a Clostridial toxinpharmaceutical composition comprises a non-protein polymer. In an aspectof this embodiment, a Clostridial toxin pharmaceutical compositioncomprises a polysaccharide. In aspects of this embodiment, a Clostridialtoxin pharmaceutical composition comprises a dextran, an inulin, astarch, a starch derivative, a hetastarch, a dextrin, a glycogen, acellulose, FICOLL, a methyl cellulose (MC), a carboxymethyl cellulose(CMC), a hydroxyethyl cellulose (HEC), a hydroxypropyl cellulose (HPC),a hydroxyethyl methyl cellulose (HEMC), or a hydroxypropyl methylcellulose (HPMC). In another aspect of this embodiment, a Clostridialtoxin pharmaceutical composition comprises a polyvinyl acetate. Inanother aspect of this embodiment, a Clostridial toxin pharmaceuticalcomposition comprises a polyvinylpyrrolidone. In aspects of thisembodiment, a Clostridial toxin pharmaceutical composition comprisesdextran 1K, dextran 4K, dextran 40K, dextran 60K, or dextran 70K. Inanother aspect of this embodiment, a Clostridial toxin pharmaceuticalcomposition comprises PVP 12, PVP 17, PVP 25, PVP 30, or PVP 90. In yetanother aspect of this embodiment, a Clostridial toxin pharmaceuticalcomposition comprises a polyethylene glycol. In an aspect of thisembodiment, a Clostridial toxin pharmaceutical composition comprises aroom temperature solid PEG. In aspects of this embodiment, a Clostridialtoxin pharmaceutical composition comprises PEG 1000, PEG 1100, PEG 1200,PEG 1300, PEG 1400, PEG 1500, PEG 1600, PEG 1700, PEG 1800, PEG 1900,PEG 2000, PEG 2100, PEG 2200, PEG 2300, PEG 2400, PEG 2500, PEG 2600,PEG 2700, PEG 2800, PEG 2900, PEG 3000, PEG 3250, PEG 3350, PEG 3500,PEG 3750, PEG 4000, PEG 4250, PEG 4500, PEG 4750, PEG 5000, PEG 5500,PEG 6000, PEG 6500, PEG 75000, PEG 7500, or PEG 8000. In another aspectof this embodiment, a Clostridial toxin pharmaceutical compositioncomprises a polyethylene imine.

It is envisioned that any amount of non-protein polymer is useful informulating a Clostridial toxin pharmaceutical compositions disclosed inthe present specification, with the proviso that a therapeuticallyeffective amount of the Clostridial toxin active ingredient is recoveredusing this non-protein polymer amount. In other aspects of thisembodiment, the amount of non-protein polymer added to the formulationis at least 0.5% (w/v), at least 1.0% (w/v), at least 2.0% (w/v), atleast 3.0% (w/v), at least 4.0% (w/v), at least 5.0% (w/v), at least6.0% (w/v), at least 7.0% (w/v), at least 8.0% (w/v), at least 9.0%(w/v), at least 10% (w/v), at least 15% (w/v), at least 20% (w/v), atleast 25% (w/v), at least 30% (w/v), or at least 35% (w/v). In otheraspects of this embodiment, the amount of non-protein polymer added tothe formulation is at most 0.5% (w/v), at most 1.0% (w/v), at most 2.0%(w/v), at most 3.0% (w/v), at most 4.0% (w/v), at most 5.0% (w/v), atmost 6.0% (w/v), at most 7.0% (w/v), at most 8.0% (w/v), at most 9.0%(w/v), at most 10% (w/v), at most 15% (w/v), at most 20% (w/v), at most25% (w/v), at most 30% (w/v), or at most 35% (w/v).

Aspects of the present pharmaceutical compositions provide, in part, asurfactant. As used hereon, the term “surfactant” refers to a natural orsynthetic amphiphilic compound. A surfactant can be non-ionic,zwitterionic, or ionic. It is envisioned that any surfactant is usefulin formulating a Clostridial toxin pharmaceutical compositions disclosedin the present specification, with the proviso that a therapeuticallyeffective amount of the Clostridial toxin active ingredient is recoveredusing this surfactant amount. Non-limiting examples of surfactantsinclude polysorbates like polysorbate 20 (TWEEN® 20), polysorbate 40(TWEEN® 40), polysorbate 60 (TWEEN® 60), polysorbate 61 (TWEEN® 61),polysorbate 65 (TWEEN® 65), polysorbate 80 (TWEEN® 80), and polysorbate81 (TWEEN® 81); poloxamers (polyethylene-polypropylene copolymers), likePoloxamer 124 (PLURONIC® L44), Poloxamer 181 (PLURONIC® L61), Poloxamer182 (PLURONIC® L62), Poloxamer 184 (PLURONIC® L64), Poloxamer 188(PLURONIC® F68), Poloxamer 237 (PLURONIC® F87), Poloxamer 338 (PLURONIC®L108), Poloxamer 407 (PLURONIC® F127), polyoxyethyleneglycol dodecylethers, like BRIJ® 30, and BRIJ® 35; 2-dodecoxyethanol (LUBROL®-PX);polyoxyethylene octyl phenyl ether (TRITON® X-100); sodium dodecylsulfate (SDS); 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS); 3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO); sucrosemonolaurate; and sodium cholate. Other non-limiting examples ofsurfactant excipients can be found in, e.g., Ansel, supra, (1999);Gennaro, supra, (2000); Hardman, supra, (2001); and Rowe, supra, (2003),each of which is hereby incorporated by reference in its entirety.

Thus in an embodiment, a Clostridial toxin pharmaceutical compositioncomprises a surfactant. In aspects of this embodiment, a Clostridialtoxin pharmaceutical composition comprises a polysorbate, a poloxamer, apolyoxyethyleneglycol dodecyl ether, 2-dodecoxyethanol, polyoxyethyleneoctyl phenyl ether, sodium dodecyl sulfate,3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate,3-[(3-Cholamidopropyl) dimethylammonio]-2-hydroxy-1-propanesulfonate,sucrose monolaurate; or sodium cholate.

It is envisioned that any amount of surfactant is useful in formulatinga Clostridial toxin pharmaceutical compositions disclosed in the presentspecification, with the proviso that a therapeutically effective amountof the Clostridial toxin active ingredient is recovered using thissurfactant amount. In aspects of this embodiment, the amount ofsurfactant added to the formulation is at least 0.5% (w/v), at least1.0% (w/v), at least 2.0% (w/v), at least 3.0% (w/v), at least 4.0%(w/v), at least 5.0% (w/v), at least 6.0% (w/v), at least 7.0% (w/v), atleast 8.0% (w/v), at least 9.0% (w/v), at least 10% (w/v), at least 15%(w/v), at least 20% (w/v), at least 25% (w/v), at least 30% (w/v), or atleast 35% (w/v). In other aspects of this embodiment, the amount ofsurfactant added to the formulation is at most 0.5% (w/v), at most 1.0%(w/v), at most 2.0% (w/v), at most 3.0% (w/v), at most 4.0% (w/v), atmost 5.0% (w/v), at most 6.0% (w/v), at most 7.0% (w/v), at most 8.0%(w/v), at most 9.0% (w/v), at most 10% (w/v), at most 15% (w/v), at most20% (w/v), at most 25% (w/v), at most 30% (w/v), or at most 35% (w/v).

In yet other aspects of this embodiment, the amount of surfactant addedto the formulation is at least 0.5% (v/v), at least 1.0% (v/v), at least2.0% (v/v), at least 3.0% (v/v), at least 4.0% (v/v), at least 5.0%(v/v), at least 6.0% (v/v), at least 7.0% (v/v), at least 8.0% (v/v), atleast 9.0% (v/v), at least 10% (v/v), at least 15% (v/v), at least 20%(v/v), at least 25% (v/v), at least 30% (v/v), or at least 35% (v/v). Inother aspects of this embodiment, the amount of surfactant added to theformulation is at most 0.5% (v/v), at most 1.0% (v/v), at most 2.0%(v/v), at most 3.0% (v/v), at most 4.0% (v/v), at most 5.0% (v/v), atmost 6.0% (v/v), at most 7.0% (v/v), at most 8.0% (v/v), at most 9.0%(v/v), at most 10% (v/v), at most 15% (v/v), at most 20% (v/v), at most25% (v/v), at most 30% (v/v), or at most 35% (v/v).

Aspects of the present pharmaceutical compositions provide, in part, anamino acid. As used hereon, the term “amino acid” refers to a moleculewith the general formula H₂NCHRCOOH, where R is an organic substitute.It is envisioned that any amino acid is useful in formulating aClostridial toxin pharmaceutical compositions disclosed in the presentspecification, with the proviso that a therapeutically effective amountof the Clostridial toxin active ingredient is recovered using this aminoacid amount. Amino acids include both the twenty standard amino acidsand non-standard amino acids. Non-limiting examples of amino acidsinclude glycine, proline, 4-hydroxyproline, serine, glutamate, alanine,lysine, sarcosine, γ-aminobutyric acid. Other non-limiting examples ofamino acids excipients can be found in, e.g., Ansel, supra, (1999);Gennaro, supra, (2000); Hardman, supra, (2001); and Rowe, supra, (2003),each of which is hereby incorporated by reference in its entirety.

Thus in an embodiment, a Clostridial toxin pharmaceutical compositioncomprises an amino acid. In aspects of this embodiment, a Clostridialtoxin pharmaceutical composition comprises a glycine, proline,4-hydroxyproline, serine, glutamate, alanine, lysine, sarcosine, orγ-aminobutyric acid.

It is envisioned that any amount of amino acid is useful in formulatinga Clostridial toxin pharmaceutical compositions disclosed in the presentspecification, with the proviso that a therapeutically effective amountof the Clostridial toxin active ingredient is recovered using this aminoacid amount. In aspects of this embodiment, the amount of amino acidadded to the formulation is at least 0.5% (w/v), at least 1.0% (w/v), atleast 2.0% (w/v), at least 3.0% (w/v), at least 4.0% (w/v), at least5.0% (w/v), at least 6.0% (w/v), at least 7.0% (w/v), at least 8.0%(w/v), at least 9.0% (w/v), at least 10% (w/v), at least 15% (w/v), atleast 20% (w/v), at least 25% (w/v), at least 30% (w/v), or at least 35%(w/v). In other aspects of this embodiment, the amount of amino acidadded to the formulation is at most 0.5% (w/v), at most 1.0% (w/v), atmost 2.0% (w/v), at most 3.0% (w/v), at most 4.0% (w/v), at most 5.0%(w/v), at most 6.0% (w/v), at most 7.0% (w/v), at most 8.0% (w/v), atmost 9.0% (w/v), at most 10% (w/v), at most 15% (w/v), at most 20%(w/v), at most 25% (w/v), at most 30% (w/v), or at most 35% (w/v).

It is envisioned that a plurality of non-protein excipients is useful informulating a Clostridial toxin pharmaceutical compositions disclosed inthe present specification, with the proviso that a therapeuticallyeffective amount of the Clostridial toxin active ingredient is recoveredusing this plurality of non-protein excipients. Thus in an embodiment, aClostridial toxin pharmaceutical composition comprises a plurality ofnon-protein excipients. In aspects of this embodiment, a Clostridialtoxin pharmaceutical composition can comprise, e.g., at least twonon-protein excipients, at least three non-protein excipients, at leastfour non-protein excipients, at least five non-protein excipients, atleast six non-protein excipients, at least seven non-protein excipientsor at least eight non-protein excipients. In other aspects of thisembodiment, a Clostridial toxin pharmaceutical composition can comprise,e.g., at most two non-protein excipients, at most three non-proteinexcipients, at most four non-protein excipients, at most fivenon-protein excipients, at most six non-protein excipients, at mostseven non-protein excipients or at most eight non-protein excipients. Inother aspects of this embodiment, a Clostridial toxin pharmaceuticalcomposition can comprise, e.g., 2-10 non-protein excipients, 2-8,non-protein excipients, 2-6 non-protein excipients, 2-4 non-proteinexcipients, 3-10 non-protein excipients, 3-8, non-protein excipients,3-6 non-protein excipients, 3-4 non-protein excipients, 4-10 non-proteinexcipients, 4-8 non-protein excipients, or 4-6 non-protein excipients.For example, a Clostridial toxin pharmaceutical composition can comprisetwo different sugars and a Clostridial toxin active ingredient, aClostridial toxin pharmaceutical composition can comprise a sugar, asurfactant and a Clostridial toxin active ingredient, a Clostridialtoxin pharmaceutical composition can comprise a non-protein polymer, asurfactant and a Clostridial toxin active ingredient, or a Clostridialtoxin pharmaceutical composition can comprise a sugar, a non-proteinpolymer, a surfactant and a Clostridial toxin active ingredient.

It is envisioned that any ratio of non-protein excipients is useful informulating a Clostridial toxin pharmaceutical compositions disclosed inthe present specification, with the proviso that a therapeuticallyeffective amount of the Clostridial toxin active ingredient is recoveredusing this excipient ratio. In aspects of this embodiment, when twonon-protein excipients are added to the formulation, the ratio of thefirst excipient to the second excipient is at least 20:1, at least 15:1,at least 10:1, at least 9:1, at least 8:1, at least 7:1, at least 6:1,at least 5:1, at least 4:1, at least 3:1, at least 2:1, at least 1:1, atleast 1:2, at least 1:3, at least 1:4, at least 1:5, at least 1:6, atleast 1:7, at least 1:8, at least 1:9, at least 1:10, at least 1:15, orat least 1:20. In other aspects of this embodiment, when threenon-protein excipients are added to the formulation, the ratio of thefirst excipient to the second excipient and third excipient is at least10:2:1, at least 9:2:1, at least 8:2:1, at least 7:2:1, at least 6:2:1,at least 5:2:1, at least 4:2:1, at least 3:2:1, at least 2:2:1, at least10:1:1, at least 9:1:1, at least 8:1:1, at least 7:1:1, at least 6:1:1,at least 5:1:1, at least 4:1:1, at least 3:1:1, at least 2:1:1, or atleast 1:1:1.

It is further envisioned that a Clostridial toxin pharmaceuticalcomposition disclosed in the present specification can optionallyinclude, without limitation, other pharmaceutically acceptablecomponents (or pharmaceutical components), including, withoutlimitation, buffers, preservatives, tonicity adjusters, salts,antioxidants, osmolality adjusting agents, emulsifying agents,sweetening or flavoring agents, and the like. Various buffers and meansfor adjusting pH can be used to prepare a pharmaceutical compositiondisclosed in the present specification, provided that the resultingpreparation is pharmaceutically acceptable. Such buffers include,without limitation, acetate buffers, citrate buffers, phosphate buffers,neutral buffered saline, phosphate buffered saline and borate buffers.It is understood that acids or bases can be used to adjust the pH of apharmaceutical composition as needed. It is envisioned that any bufferedpH level can be useful in formulating a Clostridial toxin pharmaceuticalcomposition, with the proviso that a therapeutically effective amount ofthe Clostridial toxin active ingredient is recovered using thiseffective pH level. In an aspect of this embodiment, an effective pHlevel is at least about pH 5.0, at least about pH 5.5, at least about pH6.0, at least about pH 6.5, at least about pH 7.0 or at about about pH7.5. In another aspect of this embodiment, an effective pH level is atmost about pH 5.0, at most about pH 5.5, at most about pH 6.0, at mostabout pH 6.5, at most about pH 7.0 or at most about pH 7.5. In yetanother aspect of this embodiment, an effective pH level is about pH 5.0to about pH 8.0, an effective pH level is about pH 5.0 to about pH 7.0,an effective pH level is about pH 5.0 to about pH 6.0, is about pH 5.5to about pH 8.0, an effective pH level is about pH 5.5 to about pH 7.0,an effective pH level is about pH 5.5 to about pH 5.0, is about pH 5.5to about pH 7.5, an effective pH level is about pH 5.5 to about pH pH6.5.

It is envisioned that any concentration of a buffer can be useful informulating a Clostridial toxin pharmaceutical composition, with theproviso that a therapeutically effective amount of the Clostridial toxinactive ingredient is recovered using this effective concentration ofbuffer. In aspects of this embodiment, an effective concentration ofbuffer is at least 0.1 mM, at least 0.2 mM, at least 0.3 mM, at least0.4 mM, at least 0.5 mM, at least 0.6 mM, at least 0.7 mM, at least 0.8mM, or at least 0.9 mM. In other aspects of this embodiment, aneffective concentration of buffer is at least 1.0 mM, at least 2.0 mM,at least 3.0 mM, at least 4.0 mM, at least 5.0 mM, at least 6.0 mM, atleast 7.0 mM, at least 8.0 mM, or at least 9.0 mM. In yet other aspectsof this embodiment, an effective concentration of buffer is at least 10mM, at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, atleast 60 mM, at least 70 mM, at least 80 mM, or at least 90 mM. In stillother aspects of this embodiment, an effective concentration of bufferis at least 100 mM, at least 200 mM, at least 300 mM, at least 400 mM,at least 500 mM, at least 600 mM, at least 700 mM, at least 800 mM, orat least 900 mM. In further aspects of this embodiment, an effectiveconcentration of buffer is at most 0.1 mM, at most 0.2 mM, at most 0.3mM, at most 0.4 mM, at most 0.5 mM, at most 0.6 mM, at most 0.7 mM, atmost 0.8 mM, or at most 0.9 mM. In still other aspects of thisembodiment, an effective concentration of buffer is at most 1.0 mM, atmost 2.0 mM, at most 3.0 mM, at most 4.0 mM, at most 5.0 mM, at most 6.0mM, at most 7.0 mM, at most 8.0 mM, or at most 9.0 mM. In yet otheraspects of this embodiment, an effective concentration of buffer is atmost 10 mM, at most 20 mM, at most 30 mM, at most 40 mM, at most 50 mM,at most 60 mM, at most 70 mM, at most 80 mM, or at most 90 mM. In stillother aspects of this embodiment, an effective concentration of bufferis at most 100 mM, at most 200 mM, at most 300 mM, at most 400 mM, atmost 500 mM, at most 600 mM, at most 700 mM, at most 800 mM, or at most900 mM. In still further aspects of this embodiment, an effectiveconcentration of buffer is about 0.1 mM to about 900 mM, 0.1 mM to about500 mM, 0.1 mM to about 100 mM, 0.1 mM to about 90 mM, 0.1 mM to about50 mM, 1.0 mM to about 900 mM, 1.0 mM to about 500 mM, 1.0 mM to about100 mM, 1.0 mM to about 90 mM, or 1.0 mM to about 50 mM.

Pharmaceutically acceptable antioxidants include, without limitation,sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylatedhydroxyanisole and butylated hydroxytoluene. Useful preservativesinclude, without limitation, benzalkonium chloride, chlorobutanol,thimerosal, phenylmercuric acetate, phenylmercuric nitrate, a stabilizedoxy chloro composition, such as, e.g., PURITE® and chelants, such as,e.g., DTPA or DTPA-bisamide, calcium DTPA, and CaNaDTPA-bisamide.Tonicity adjustors useful in a pharmaceutical composition include,without limitation, salts such as, e.g., sodium chloride and potassiumchloride. The pharmaceutical composition may be provided as a salt andcan be formed with many acids, including but not limited to,hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc.Salts tend to be more soluble in aqueous or other protonic solvents thanare the corresponding free base forms. It is understood that these andother substances known in the art of pharmacology can be included in apharmaceutical composition useful in the invention. Other non-limitingexamples of pharmacologically acceptable components can be found in,e.g., Ansel, supra, (1999); Gennaro, supra, (2000); Hardman, supra,(2001); and Rowe, supra, (2003), each of which is hereby incorporated byreference in its entirety.

It is envisioned that any concentration of a salt can be useful informulating a Clostridial toxin pharmaceutical composition, with theproviso that a therapeutically effective amount of the Clostridial toxinactive ingredient is recovered using this effective concentration ofsalt. In aspects of this embodiment, an effective concentration of saltis at least 0.1 mM, at least 0.2 mM, at least 0.3 mM, at least 0.4 mM,at least 0.5 mM, at least 0.6 mM, at least 0.7 mM, at least 0.8 mM, orat least 0.9 mM. In other aspects of this embodiment, an effectiveconcentration of salt is at least 1.0 mM, at least 2.0 mM, at least 3.0mM, at least 4.0 mM, at least 5.0 mM, at least 6.0 mM, at least 7.0 mM,at least 8.0 mM, or at least 9.0 mM. In yet other aspects of thisembodiment, an effective concentration of salt is at least 10 mM, atleast 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60mM, at least 70 mM, at least 80 mM, or at least 90 mM. In still otheraspects of this embodiment, an effective concentration of salt is atleast 100 mM, at least 200 mM, at least 300 mM, at least 400 mM, atleast 500 mM, at least 600 mM, at least 700 mM, at least 800 mM, or atleast 900 mM. In further aspects of this embodiment, an effectiveconcentration of salt is at most 0.1 mM, at most 0.2 mM, at most 0.3 mM,at most 0.4 mM, at most 0.5 mM, at most 0.6 mM, at most 0.7 mM, at most0.8 mM, or at most 0.9 mM. In still other aspects of this embodiment, aneffective concentration of salt is at most 1.0 mM, at most 2.0 mM, atmost 3.0 mM, at most 4.0 mM, at most 5.0 mM, at most 6.0 mM, at most 7.0mM, at most 8.0 mM, or at most 9.0 mM. In yet other aspects of thisembodiment, an effective concentration of salt is at most 10 mM, at most20 mM, at most 30 mM, at most 40 mM, at most 50 mM, at most 60 mM, atmost 70 mM, at most 80 mM, or at most 90 mM. In still other aspects ofthis embodiment, an effective concentration of salt is at most 100 mM,at most 200 mM, at most 300 mM, at most 400 mM, at most 500 mM, at most600 mM, at most 700 mM, at most 800 mM, or at most 900 mM. In stillfurther aspects of this embodiment, an effective concentration of saltis about 0.1 mM to about 900 mM, 0.1 mM to about 500 mM, 0.1 mM to about100 mM, 0.1 mM to about 90 mM, 0.1 mM to about 50 mM, 1.0 mM to about900 mM, 1.0 mM to about 500 mM, 1.0 mM to about 100 mM, 1.0 mM to about90 mM, or 1.0 mM to about 50 mM.

A pharmaceutical compositions disclosed in the present specificationgenerally is administered as a pharmaceutical acceptable compositioncomprising a botulinum toxin active ingredient. As used herein, the term“pharmaceutically acceptable” means any molecular entity or compositionthat does not produce an adverse, allergic or other untoward or unwantedreaction when administered to an individual. As used herein, the term“pharmaceutically acceptable composition” is synonymous with“pharmaceutical composition” and means a therapeutically effectiveconcentration of an active ingredient, such as, e.g., any of theClostridial toxin active ingredients disclosed in the presentspecification. A pharmaceutical composition comprising a Clostridialtoxin active ingredient is useful for medical and veterinaryapplications. A pharmaceutical composition may be administered to apatient alone, or in combination with other supplementary activeingredients, agents, drugs or hormones.

Aspects of the present pharmaceutical compositions provide, in part,recovered potency of a pharmaceutical composition. As used hereon, theterm “recovered potency” is synonymous with “recovered activity” and,when used in reference to a solid-form Clostridial toxin pharmaceuticalcomposition, refers to the percentage calculated by dividing the potencyof the Clostridial toxin active ingredient in the stored reconstitutionformulation by the potency of the active Clostridial toxin ingredientdetermined prior to its addition into the test solution. When used inreference to an aqueous-form Clostridial toxin pharmaceuticalcomposition, “recovered potency” refers to the percentage calculated bydividing the potency of the Clostridial toxin active ingredient in thestored formulation by the potency of the active Clostridial toxiningredient determined prior to its addition into the test solution. Themaximum theoretical recovered potency is 100%. As used herein, the term“potency” refers to the level of biological activity exhibited by aClostridial toxin active ingredient as measured by, e.g., a mousebioassay or an in vitro Clostridial toxin light chain activity assay. Asa non-limiting example, with respect to a solid-form Clostridial toxinpharmaceutical composition, a recovery of 60% means that the potency ofthe Clostridial toxin active ingredient after reconstitution was 60% ofthe potency of the Clostridial toxin active ingredient prior to itsaddition to the formulation. As another non-limiting example, withrespect to an aqueous-form Clostridial toxin pharmaceutical composition,a recovery of 50% means that the potency of the Clostridial toxin activeingredient after storage was 50% of the potency of the Clostridial toxinactive ingredient prior to its addition to the formulation.

It is envisioned that any level of recovered potency is useful informulating a Clostridial toxin pharmaceutical compositions disclosed inthe present specification, with the proviso that a therapeuticallyeffective amount of the Clostridial toxin active ingredient is present.Thus, in an embodiment, a Clostridial toxin pharmaceutical compositiondisclosed in the present specification exhibits a recovered potency ofat least 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, or at least 100%.In another embodiment, a Clostridial toxin pharmaceutical compositiondisclosed in the present specification exhibits a recovered potency ofat most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most60%, at most 70%, at most 80%, at most 90%, or at most 100%. In yetanother embodiment, a Clostridial toxin pharmaceutical compositiondisclosed in the present specification exhibits a recovered potency ofabout 20% to about 100%, about 20% to about 90%, about 20% to about 80%,about 20% to about 70%, about 20% to about 60%, or about 20% to about50%. In still another embodiment, a Clostridial toxin pharmaceuticalcomposition disclosed in the present specification exhibits a recoveredpotency of about 40% to about 100%, about 40% to about 90%, about 40% toabout 80%, about 40% to about 70%, about 40% to about 60%, or about 40%to about 50%.

Aspects of the present pharmaceutical compositions provide, in part, apharmaceutical composition form. As used herein, the term“pharmaceutical composition form” refers to whether the pharmaceuticalcomposition is processed into a solid form or aqueous form. Processing aformulation of a pharmaceutical composition into a solid form can beachieved by, e.g., lyophilization (freeze-drying) or vacuum-drying.Processing a formulation of a pharmaceutical composition into an aqueousform can simply be achieved during the compounding stage by the additionof a solute that dissolves or suspends solid excipients to form asolution. Thus, in an embodiment, a Clostridial toxin pharmaceuticalcomposition is in a solid form. In another embodiment, a Clostridialtoxin pharmaceutical composition is in an aqueous form.

Aspects of the present pharmaceutical compositions provide, in part,storage condition of a pharmaceutical composition. As used hereon, theterm “storage condition of a pharmaceutical composition” refers to thelocation a pharmaceutical composition is stored while in its solid formbefore reconstitution with an appropriate solution prior toadministration. It is envisioned that any storage condition is usefulfor storing a Clostridial toxin pharmaceutical compositions disclosed inthe present specification, with the proviso that a therapeuticallyeffective amount of the Clostridial toxin active ingredient is recoveredupon reconstitution with the appropriate solution. In an embodiment, aClostridial toxin pharmaceutical composition disclosed in the presentspecification is stored at ambient temperature. In aspects of thisembodiment, a Clostridial toxin pharmaceutical composition disclosed inthe present specification is stored at an ambient temperature of atleast 16° C., at least 18° C., at least 20° C., or at least 22° C. Inother aspects of this embodiment, a Clostridial toxin pharmaceuticalcomposition disclosed in the present specification is stored at anambient temperature of at most 16° C., at most 18° C., at most 20° C.,or at most 22° C. In yet other aspects of this embodiment, a Clostridialtoxin pharmaceutical composition disclosed in the present specificationis stored at an ambient temperature of about 16° C. to about 24° C., atabout 16° C. to about 22° C., at about 16° C. to about 20° C., or atabout 18° C. to about 24° C. In another embodiment, a Clostridial toxinpharmaceutical composition disclosed in the present specification isstored at a temperature below freezing. In aspects of this embodiment, aClostridial toxin pharmaceutical composition disclosed in the presentspecification is stored at a temperature of at least 0° C., at least−20° C., at least −70° C., or at least −120° C. In other aspects of thisembodiment, a Clostridial toxin pharmaceutical composition disclosed inthe present specification is stored at a temperature of at most 0° C.,at most −20° C., at most −70° C., or at most −120° C. In yet otheraspects of this embodiment, a Clostridial toxin pharmaceuticalcomposition disclosed in the present specification is stored at atemperature of at about 0° C. to about −20° C., at about −5° C. to about−20° C., at about 0° C. to about −15° C., at about −5° C. to about −15°C., at about 0° C. to about −70° C., at about −20° C. to about −70° C.,or at about −20° C. to about −120° C.

Aspects of the present pharmaceutical compositions provide, in part, aClostridial toxin active ingredient that is stable. For purposes of thepresent Clostridial toxin pharmaceutical compositions, a Clostridialtoxin active ingredient is stable when the recovered potency of theactive ingredient when stored for a certain period of time is at least70% of the initial recovered potency for that active ingredient. Forexample, a Clostridial toxin active ingredient is stable when theClostridial toxin pharmaceutical composition containing that Clostridialtoxin active ingredient demonstrates, e.g., an initial recovered potencyof 100% and a recovered potency of at least 70% when tested one yearlater, an initial recovered potency of 90% and a recovered potency of atleast 63% when tested one year later, an initial recovered potency of80% and a recovered potency of at least 56% when tested one year later,an initial recovered potency of 70% and a recovered potency of at least49% when tested one year later, or an initial recovered potency of 60%and a recovered potency of at least 42% when tested one year later.

Aspects of the Clostridial toxin pharmaceutical compositions disclosedin the present specification can also be described as follows:

-   1. A citrate-buffered, animal-protein free, solid-form Clostridial    toxin pharmaceutical composition comprising a Clostridial toxin    active ingredient and an effective amount of sucrose, wherein the    composition is buffered to about pH 5.5, and wherein the Clostridial    toxin active ingredient is stable for at least one-year when stored    at either ambient or below freezing temperatures.-   2. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient and an effective amount of lactose, wherein the    Clostridial toxin active ingredient is stable for at least one-year    when stored at either ambient or below freezing temperatures.-   3. A buffered, animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient and an effective amount of lactose, wherein the    composition is buffered to about pH 5.5, and wherein the Clostridial    toxin active ingredient is stable for at least one-year when stored    at either ambient or below freezing temperatures.-   4. A citrate-buffered, animal-protein free, solid-form Clostridial    toxin pharmaceutical composition comprising a Clostridial toxin    active ingredient and an effective amount of lactose, wherein the    composition is buffered to about pH 6.5, and wherein the Clostridial    toxin active ingredient is stable for at least one-year when stored    at below freezing temperatures.-   5. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient and an effective amount of lactose in sodium chloride    solution, wherein the Clostridial toxin active ingredient is stable    for at least one-year when stored at below freezing temperatures.-   6. A phosphate-buffered, animal-protein free, solid-form Clostridial    toxin pharmaceutical composition comprising a Clostridial toxin    active ingredient and an effective amount of dextran 3K, wherein the    composition is buffered to about pH 6.5, and wherein the Clostridial    toxin active ingredient is stable for at least one-year when stored    at below freezing temperatures.-   7. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient and an effective amount of PVP 17, wherein the    Clostridial toxin active ingredient is stable for at least one-year    when stored at below freezing temperatures.-   8. A buffered, animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient and an effective amount of PVP 17, wherein the    composition is buffered to about pH 5.5 to about pH 6.5, and wherein    the Clostridial toxin active ingredient is stable for at least    one-year when stored at below freezing temperatures.-   9. A citrate-buffered, animal-protein free, solid-form Clostridial    toxin pharmaceutical composition comprising a Clostridial toxin    active ingredient and an effective amount of PVP 17, wherein the    composition is buffered to about pH 5.5, and wherein the Clostridial    toxin active ingredient is stable for at least one-year when stored    at either ambient or below freezing temperatures.-   10. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of PVP 17, and an effective amount    of sodium chloride, wherein the Clostridial toxin active ingredient    is stable for at least one-year when stored at below freezing    temperatures.-   11. A citrate-buffered, animal-protein free, solid-form Clostridial    toxin pharmaceutical composition comprising a Clostridial toxin    active ingredient and an effective amount of PEG 3350, wherein the    composition is buffered to about pH 5.5 to about pH 6.5, and wherein    the Clostridial toxin active ingredient is stable for at least    one-year when stored at either ambient or below freezing    temperatures.-   12. A histidine-buffered, animal-protein free, solid-form    Clostridial toxin pharmaceutical composition comprising a    Clostridial toxin active ingredient and an effective amount of PEG    3350, wherein the composition is buffered to about pH 5.5 to about    pH 6.5, and wherein the Clostridial toxin active ingredient is    stable for at least one-year when stored at either ambient or below    freezing temperatures.-   13. A citrate-buffered, animal-protein free, solid-form Clostridial    toxin pharmaceutical composition comprising a Clostridial toxin    active ingredient and an effective amount of Poloxamer 188, wherein    the composition is buffered to about pH 5.5, and wherein the    Clostridial toxin active ingredient is stable for at least one-year    when stored at either ambient or below freezing temperatures.-   14. A citrate-buffered, animal-protein free, solid-form Clostridial    toxin pharmaceutical composition comprising a Clostridial toxin    active ingredient and an effective amount of Poloxamer 188, wherein    the composition is buffered to about pH 5.5 to about pH 6.5, and    wherein the Clostridial toxin active ingredient is stable for at    least one-year when stored at below freezing temperatures.-   15. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of lactose and an effective amount    of sucrose, wherein the Clostridial toxin active ingredient is    stable for at least one-year when stored at either ambient or below    freezing temperatures.-   16. A buffered, animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of lactose and an effective amount    of sucrose, wherein the composition is buffered to about pH 5.5, and    wherein the Clostridial toxin active ingredient is stable for at    least one-year when stored at either ambient or below freezing    temperatures.-   17. A phosphate-buffered, animal-protein free, solid-form    Clostridial toxin pharmaceutical composition comprising a    Clostridial toxin active ingredient, effective amount of lactose and    an effective amount of sucrose, wherein the composition is buffered    to about pH 6.5, and wherein the Clostridial toxin active ingredient    is stable for at least one-year when stored at below freezing    temperatures.-   18. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of lactose, an effective amount of    sucrose and an effective amount of sodium chloride, wherein the    Clostridial toxin active ingredient is stable for at least one-year    when stored at either ambient or below freezing temperatures.-   19. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of sucrose and an effective amount    of PVP 17, wherein the Clostridial toxin active ingredient is stable    for at least one-year when stored at below freezing temperatures.-   20. A buffered, animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of sucrose and an effective amount    of PVP 17, wherein the composition is buffered to about pH 5.5 to    about pH 6.5, and wherein the Clostridial toxin active ingredient is    stable for at least one-year when stored at below freezing    temperatures.-   21. A citrate-buffered, animal-protein free, solid-form Clostridial    toxin pharmaceutical composition comprising a Clostridial toxin    active ingredient, an effective amount of sucrose and an effective    amount of PVP 17, wherein the composition is buffered to about pH    5.5, and wherein the Clostridial toxin active ingredient is stable    for at least one-year when stored at either ambient or below    freezing temperatures.-   22. A phosphate-buffered, animal-protein free, solid-form    Clostridial toxin pharmaceutical composition comprising a    Clostridial toxin active ingredient, an effective amount of sucrose    and an effective amount of PVP 17, wherein the composition is    buffered to about pH 5.5 to about pH 6.5, and wherein the    Clostridial toxin active ingredient is stable for at least one-year    when stored at either ambient or below freezing temperatures.-   23. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of sucrose, an effective amount of    PVP 17 and an effective amount of sodium chloride, wherein the    Clostridial toxin active ingredient is stable for at least one-year    when stored at either ambient or below freezing temperatures.-   24. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of sucrose and an effective amount    of PEG 3350, wherein the Clostridial toxin active ingredient is    stable for at least one-year when stored at either ambient or below    freezing temperatures.-   25. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of lactose and an effective amount    of PVP 17, wherein the Clostridial toxin active ingredient is stable    for at least one-year when stored at below freezing temperatures.-   26. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of lactose and an effective amount    of PEG 3350, wherein the Clostridial toxin active ingredient is    stable for at least one-year when stored at either ambient or below    freezing temperatures.-   26. A citrate-buffered, animal-protein free, solid-form Clostridial    toxin pharmaceutical composition comprising a Clostridial toxin    active ingredient, an effective amount of lactose and an effective    amount of PEG 3350, wherein the composition is buffered to about pH    5.5, and wherein the Clostridial toxin active ingredient is stable    for at least one-year when stored at either ambient or below    freezing temperatures.-   27. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of sucrose and an effective amount    of Poloxamer 188, wherein the Clostridial toxin active ingredient is    stable for at least one-year when stored at below freezing    temperatures.-   28. A buffered, animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of sucrose and an effective amount    of Poloxamer 188, wherein the composition is buffered to about pH    5.5 to about pH 6.5, and wherein the Clostridial toxin active    ingredient is stable for at least one-year when stored at either    ambient or below freezing temperatures.-   29. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of sucrose, an effective amount of    Poloxamer 188, and an effective amount of sodium chloride, wherein    the Clostridial toxin active ingredient is stable for at least    one-year when stored at either ambient or below freezing    temperatures.-   30. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of sucrose and an effective amount    of polysorbate 80, wherein the Clostridial toxin active ingredient    is stable for at least one-year when stored at below freezing    temperatures.-   31. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of lactose and an effective amount    of Poloxamer 188, wherein the Clostridial toxin active ingredient is    stable for at least one-year when stored at below freezing    temperatures.-   32. A buffered, animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of lactose and an effective amount    of Poloxamer 188, wherein the composition is buffered to about pH    5.5 to an about pH 6.5, and wherein the Clostridial toxin active    ingredient is stable for at least one-year when stored at either    ambient or below freezing temperatures.-   33. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of lactose, an effective amount of    Poloxamer 188, and an effective amount of sodium chloride, wherein    the Clostridial toxin active ingredient is stable for at least    one-year when stored at either ambient or below freezing    temperatures.-   34. A buffered, animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of Dextran 3K and an effective    amount of PEG 3350, wherein the composition is buffered to about pH    5.5 to about pH 6.5, and wherein the Clostridial toxin active    ingredient is stable for at least one-year when stored at below    freezing temperatures.-   35. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of PVP 17 and an effective amount of    PEG 3350, wherein the Clostridial toxin active ingredient is stable    for at least one-year when stored at below freezing temperatures.-   36. A buffered, animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of PVP 17 and an effective amount of    PEG 3350, wherein the composition is buffered to about pH 5.5 to    about pH 6.5, and wherein the Clostridial toxin active ingredient is    stable for at least one-year when stored at below freezing    temperatures.-   37. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of Dextran 3K and an effective    amount of Poloxamer 188, wherein the Clostridial toxin active    ingredient is stable for at least one-year when stored at below    freezing temperatures.-   38. A buffered, animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of Dextran 3K and an effective    amount of Poloxamer 188, wherein the composition is buffered to    about pH 5.5 to about pH 6.5, and wherein the Clostridial toxin    active ingredient is stable for at least one-year when stored at    below freezing temperatures.-   39. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of Dextran 40K and an effective    amount of Poloxamer 188, wherein the Clostridial toxin active    ingredient is stable for at least one-year when stored at below    freezing temperatures.-   40. A buffered, animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of Dextran 40K and an effective    amount of Poloxamer 188, wherein the composition is buffered to    about pH 5.5 to about pH 6.5, and wherein the Clostridial toxin    active ingredient is stable for at least one-year when stored at    below freezing temperatures.-   41. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of PVP 17 and an effective amount of    Poloxamer 188, wherein the Clostridial toxin active ingredient is    stable for at least one-year when stored at below freezing    temperatures.-   42. A buffered, animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of PVP 17 and an effective amount of    Poloxamer 188, wherein the composition is buffered to about pH 5.5    to about pH 6.5, and wherein the Clostridial toxin active ingredient    is stable for at least one-year when stored at below freezing    temperatures.-   43. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of PVP 17, an effective amount of    Poloxamer 188, and an effective amount of sodium chloride, wherein    the Clostridial toxin active ingredient is stable for at least    one-year when stored at below freezing temperatures.-   44. A citrate-buffered, animal-protein free, solid-form Clostridial    toxin pharmaceutical composition comprising a Clostridial toxin    active ingredient, an effective amount of PEG 3350 and an effective    amount of Poloxamer 188, wherein the composition is buffered to    about pH 5.5 to about pH 6.5, and wherein the Clostridial toxin    active ingredient is stable for at least one-year when stored at    below freezing temperatures.-   45. A phosphate-buffered, animal-protein free, solid-form    Clostridial toxin pharmaceutical composition comprising a    Clostridial toxin active ingredient, an effective amount of PEG 3350    and an effective amount of Poloxamer 188, wherein the composition is    buffered to about pH 5.5, and wherein the Clostridial toxin active    ingredient is stable for at least one-year when stored at below    freezing temperatures.-   46. A histidine-buffered, animal-protein free, solid-form    Clostridial toxin pharmaceutical composition comprising a    Clostridial toxin active ingredient, an effective amount of PEG 3350    and an effective amount of Poloxamer 188, wherein the composition is    buffered to about pH 5.5 to about pH 6.5, and wherein the    Clostridial toxin active ingredient is stable for at least one-year    when stored at either ambient or below freezing temperatures.-   47. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of PVP 17 and an effective amount of    Polysorbate 80, wherein the Clostridial toxin active ingredient is    stable for at least one-year when stored at below freezing    temperatures.-   48. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of sucrose, an effective amount of    PVP 17 and an effective amount of Poloxamer 188, wherein the    Clostridial toxin active ingredient is stable for at least one-year    when stored at below freezing temperatures.-   49. A buffered, animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of sucrose, an effective amount of    PVP 17 and an effective amount of Poloxamer 188, wherein the    composition is buffered to about pH 5.5 to about pH 6.5, and wherein    the Clostridial toxin active ingredient is stable for at least    one-year when stored at below freezing temperatures.-   50. A phosphate-buffered, animal-protein free, solid-form    Clostridial toxin pharmaceutical composition comprising a    Clostridial toxin active ingredient, an effective amount of sucrose,    an effective amount of PVP 17 and an effective amount of Poloxamer    188, wherein the composition is buffered to about pH 5.5 to about pH    6.5, and wherein the Clostridial toxin active ingredient is stable    for at least one-year when stored at either ambient or below    freezing temperatures.-   51. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of sucrose, an effective amount of    PVP 17, an effective amount of Poloxamer 188, and an effective    amount of sodium chloride, wherein the Clostridial toxin active    ingredient is stable for at least one-year when stored at either    ambient or below freezing temperatures.-   52. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of sucrose, an effective amount of    lactose and an effective amount of Poloxamer 188, wherein the    Clostridial toxin active ingredient is stable for at least one-year    when stored at either ambient or below freezing temperatures.-   53. A buffered, animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of sucrose, an effective amount of    lactose and an effective amount of Poloxamer 188, wherein the    composition is buffered to about pH 5.5 to about pH 6.5, and wherein    the Clostridial toxin active ingredient is stable for at least    one-year when stored at either ambient or below freezing    temperatures.-   54. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of sucrose, an effective amount of    PVP 17 and an effective amount of PEG 3350, wherein the Clostridial    toxin active ingredient is stable for at least one-year when stored    at either ambient or below freezing temperatures.-   55. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of lactose, an effective amount of    PEG 3350 and an effective amount of Poloxamer 188, wherein the    Clostridial toxin active ingredient is stable for at least one-year    when stored at either ambient or below freezing temperatures.-   56. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of Dextran 3K, an effective amount    of PEG 3350 and an effective amount of Poloxamer 188, wherein the    Clostridial toxin active ingredient is stable for at least one-year    when stored at either ambient or below freezing temperatures.-   57. A buffered, animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of Dextran 3K, an effective amount    of PEG 3350 and an effective amount of Poloxamer 188, wherein the    composition is buffered to about pH 5.5 to about pH 6.5, and wherein    the Clostridial toxin active ingredient is stable for at least    one-year when stored at either ambient or below freezing    temperatures.-   57. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of PVP 17, an effective amount of    PEG 3350 and an effective amount of Poloxamer 188, wherein the    Clostridial toxin active ingredient is stable for at least one-year    when stored at either ambient or below freezing temperatures.-   58. A buffered, animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of PVP 17, an effective amount of    PEG 3350 and an effective amount of Poloxamer 188, wherein the    composition is buffered to about pH 5.5 to about pH 6.5, and wherein    the Clostridial toxin active ingredient is stable for at least    one-year when stored at either ambient or below freezing    temperatures.-   58. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of PVP 17, an effective amount of    glycine and an effective amount of Poloxamer 188, wherein the    Clostridial toxin active ingredient is stable for at least one-year    when stored at either ambient or below freezing temperatures.-   59. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of a sugar excipient and an    effective amount of surfactant excipient.-   60. The composition according to 59, wherein the sugar excipient is    a monosaccharide, a disaccharide or a trisaccharide.-   61. The composition according to 59, wherein the surfactant    excipient is a poloxamer, a polysorbate, a polyoxyethylene glycol    dodecyl ether, or a polyoxyethylene octyl phenyl ether.-   62. The composition according to 59, wherein the Clostridial toxin    active ingredient is stable for at least one-year when stored at    either ambient or below freezing temperatures.-   63. The composition according to 59, wherein the composition is    buffered to about pH 5.5 to about pH 6.5.-   64. The composition according to 63, wherein the composition is    buffered using a citrate buffer, a phosphate buffer or a histidine    buffer.-   65. The composition according to 59, wherein the composition further    comprises an effective amount of sodium chloride.-   66. The composition according to 59, wherein the composition further    comprises an effective amount of a non-protein polymer excipient.-   67. The composition according to 66, wherein the non-protein polymer    excipient is a dextran, a polyethylene glycol, a polyethylene imine,    a polyvinyl pyrrolidone, a polyvinyl acetate, an inulin, a starch,    or a starch derivative.-   68. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of a non-protein polymer excipient    and an effective amount of surfactant excipient.-   69. The composition according to 68, wherein the non-protein polymer    excipient is a dextran, a polyethylene glycol, a polyethylene imine,    a polyvinyl pyrrolidone, a polyvinyl acetate, an inulin, a starch,    or a starch derivative.-   70. The composition according to 68, wherein the surfactant    excipient is a poloxamer, a polysorbate, a polyoxyethylene glycol    dodecyl ether, or a polyoxyethylene octyl phenyl ether.-   71. The composition according to 68, wherein the Clostridial toxin    active ingredient is stable for at least one-year when stored at    either ambient or below freezing temperatures.-   72. The composition according to 68, wherein the composition is    buffered to about pH 5.5 to about pH 6.5.-   73. The composition according to 72, wherein the composition is    buffered using a citrate buffer, a phosphate buffer or a histidine    buffer.-   74. The composition according to 68, wherein the composition further    comprises an effective amount of sodium chloride.-   75. An animal-protein free, solid-form Clostridial toxin    pharmaceutical composition comprising a Clostridial toxin active    ingredient, an effective amount of a first non-protein polymer    excipient, an effective amount of a second non-protein polymer    excipient, and an effective amount of surfactant excipient.-   76. The composition according to 75, wherein the first non-protein    polymer excipient is a dextran, a polyethylene glycol, a    polyethylene imine, a polyvinyl pyrrolidone, a polyvinyl acetate, an    inulin, a starch, or a starch derivative.-   77. The composition according to 75, wherein the second non-protein    polymer excipient is a dextran, a polyethylene glycol, a    polyethylene imine, a polyvinyl pyrrolidone, a polyvinyl acetate, an    inulin, a starch, or a starch derivative.-   78. The composition according to 75, wherein the surfactant    excipient is a poloxamer, a polysorbate, a polyoxyethylene glycol    dodecyl ether, or a polyoxyethylene octyl phenyl ether.-   79. The composition according to 75, wherein the Clostridial toxin    active ingredient is stable for at least one-year when stored at    either ambient or below freezing temperatures.-   80. The composition according to 75, wherein the composition is    buffered to about pH 5.5 to about pH 6.5.-   81. The composition according to 80, wherein the composition is    buffered using a citrate buffer, a phosphate buffer or a histidine    buffer.-   82. The composition according to 75, wherein the composition further    comprises an effective amount of sodium chloride.-   83. The composition according to 1-82, wherein the Clostridial toxin    active ingredient is a Clostridial toxin complex, a Clostridial    toxin, a modified Clostridial toxin or a re-targeted Clostridial    toxin.-   84. The composition according to 83, wherein the Clostridial toxin    complex is a BoNT/A complex, a BoNT/B complex, a BoNT/C, complex, a    BoNT/D complex, a BoNT/E complex, a BoNT/F complex, a BoNT/G    complex, a TeNT complex, a BaNT complex, or a BuNT complex.-   85. The composition according to 83, wherein the Clostridial toxin    complex is a 900-kDa BoNT/A complex, a 500-kDa BoNT/A complex, a    300-kDa BoNT/A complex, a 500-kDa BoNT/B complex, a 500-kDa BoNT/C1    complex, a 500-kDa BoNT/D complex, a 300-kDa BoNT/D complex, a    300-kDa BoNT/E complex, or a 300-kDa BoNT/F complex.-   86. The composition according to 83, wherein the Clostridial toxin    is a BoNT/A, a BoNT/B, a BoNT/C₁, a BoNT/D, a BoNT/E, a BoNT/F, a    BoNT/G, a TeNT, a BaNT, or a BuNT.-   87. The composition according to 83, wherein the BoNT/A is a    BoNT/A1, a BoNT/A2, a BoNT/A3, a BoNT/A4, or a BoNT/A5.-   88. The composition according to 83, wherein the re-targeted    Clostridial toxin is a re-targeted BoNT/A, a re-targeted BoNT/B, a    re-targeted BoNT/C1, a re-targeted BoNT/D, a re-targeted BoNT/E, a    re-targeted BoNT/F, a re-targeted BoNT/G, a re-targeted TeNT, a    re-targeted BaNT, or a re-targeted BuNT.-   89. The composition according to 83, wherein the re-targeted    Clostridial toxin comprises an opioid targeting moiety, a tachykinin    targeting moiety, a melanocortin targeting moiety, a granin    targeting moiety, a Neuropeptide Y related peptide targeting moiety,    a neurohormone targeting moiety, a neuroregulatory cytokine    targeting moiety, a kinin peptide targeting moiety, a fibroblast    growth factor targeting moiety, a nerve growth factor targeting    moiety, an insulin growth factor targeting moiety, an epidermal    growth factor targeting moiety, a vascular endothelial growth factor    targeting moiety, a brain derived neurotrophic factor targeting    moiety, a growth derived neurotrophic factor targeting moiety, a    neurotrophin targeting moiety, a head activator peptide targeting    moiety, a neurturin targeting moiety, a persephrin targeting moiety,    an artemin targeting moiety, a transformation growth factor β    targeting moiety, a bone morphogenic protein targeting moiety, a    growth differentiation factor targeting moiety, an activin targeting    moiety, a glucagon like hormone targeting moiety, a pituitary    adenylate cyclase activating peptide targeting moiety, a growth    hormone-releasing hormone targeting moiety, vasoactive intestinal    peptide targeting moiety, a gastric inhibitory polypeptide targeting    moiety, a calcitonin-related peptidesvisceral gut peptide targeting    moiety, or a PAR peptide targeting moiety.-   90. The composition according to 89, wherein the opioid targeting    moiety is an enkephalin, an endomorphin, an endorphin, a dynorphin,    a nociceptin or a hemorphin.-   91. The composition according to 89, wherein the tachykinin    targeting moiety is a Substance P, a neuropeptide K, a neuropeptide    gamma, a neurokinin A, a neurokinin B, a hemokinin or a endokinin.

EXAMPLES

The following examples set forth specific embodiments of the presentClostridial toxin pharmaceutical compositions and are not intended tolimit the scope of the invention.

Example 1 Non-Protein Stabilized Formulations—One Excipient

Experiments were carried out to determine the effects of formulationscomprising a single non-protein excipient on Clostridial toxin activeingredient recovery after reconstitution. The non-protein excipientstested were added separately or in combination with the listed buffersor salts (Table 2). All of the formulations were compounded,lyophilized, reconstituted and potency assessed in the same manner, andwith the same Clostridial toxin active ingredient used in eachformulation, except that each formulation was prepared with differentnon-protein excipient or with different amounts of the non-proteinexcipient.

Formulations were compounded by first adding the indicated amount of thenon-protein excipient(s) to sterile water to form a solution. Next theClostridial toxin active ingredient was added to the solution to producethe formulation. The Clostridial toxin active ingredient added was about150 units of a 900-kDaBoNT/A complex, about 150 units of a 150 kDaBoNT/A, or about 250 ng of a 100 kDa re-targeted BoNT/A, where themodification was the substitution of the BoNT/A binding domain with anopioid ligand, see e.g., Steward, L. E. et al., Modified ClostridialToxins with Enhanced Translocation Capabilities and Altered TargetingActivity For Non-Clostridial Toxin Target Cells, U.S. patent applicationSer. No. 11/776,075 (Jul. 11, 2007); Dolly, J. O. et al., ActivatableClostridial Toxins, U.S. patent application Ser. No. 11/829,475 (Jul.27, 2007); Foster, K. A. et al., Fusion Proteins, International PatentPublication WO 2006/059093 (Jun. 8, 2006); and Foster, K. A. et al.,Non-Cytotoxic Protein Conjugates, International Patent Publication WO2006/059105 (Jun. 8, 2006), each of which is incorporated by referencein its entirety. The formulations were processed into solid forms(either by lyophilization or vacuum drying), stored for a specifiedperiod of time (about one-day, at least three months or at least oneyear), reconstitution with either sterile water or a specified buffer,and then assayed to determine the recovered potency of the Clostridialtoxin active ingredient.

To determine the recovered potency of a Clostridial toxin, Clostridialtoxin complex or modified Clostridial toxin, the reconstitutedformulation was assayed by a mouse LD₅₀ bioassay. For each reconstitutedformulation, a minimum of six serial dilutions at 1.33 dose intervalswere prepared in normal saline and typically five or six mice (femaleSwiss Weber weighing between 17-22 grams) were used in each dosagegroup. The mice were injected intraperitoneally into the lower rightabdomen and the death rates over the ensuing 72 hours for each dilutionwere recorded. The dilutions were prepared so that the most concentrateddilution produces a death rate of at least 80% of the mice injected, andthe least concentration dilution produces a death rate no greater than20% of the mice injected. A minimum of four dilutions must fall withinthe monotone decreasing range of the death rates, i.e., the two largestand the two smallest rates must be decreasing (not equivalent). Themonotone decreasing range commences with a death rate of no less than80%. Two reference standard assays are carried out concurrently. Thedilution at which 50% of the mice die within the three day postinjection observation period is defined as a dilution which comprisesone unit (1 U) of the botulinum toxin. The mouse LD₅₀ bioassay providesa determination of the potency of a Clostridial toxin, Clostridial toxincomplex or modified Clostridial toxin in terms of its mouse 50% lethaldose or “LD₅₀.” Thus, one unit (U) of a Clostridial toxin, Clostridialtoxin complex or modified Clostridial toxin is defined as the amount oftoxin which upon intraperitoneal injection killed 50% of the miceinjected, i.e., LD₅₀.

Recovery is expressed as a percentage and is calculated by dividing thepotency of the Clostridial toxin active ingredient in the storedreconstitution formulation by the potency of the active Clostridialtoxin ingredient determined prior to its addition into the testsolution. Thus, for example, a recovery of 60% means that the potency ofthe Clostridial toxin active ingredient after reconstitution was 60% ofthe potency of the Clostridial toxin active ingredient prior to itsaddition to the formulation. The maximum theoretical recovered potencyis 100%. The results show that, in general, a Clostridial toxinpharmaceutical composition comprising a Clostridial toxin complex waspoorly stabilized when the formulation comprised a single non-proteinexcipient (Table 2).

When the single excipient used was a sugar, only the disaccharidelactose exhibited any degree of initial recovered potency, showing about15% to about 41% recovery of the Clostridial toxin active ingredientwhen about 10 mg to about 50 mg of lactose was added (about 1% (w/v) toabout 5% (w/v)) (Table 2). Furthermore, although exhibiting recovery,the test formulations containing lactose as the single excipient did notappear very stable after one year in storage since recovered potency wasnot detected at this time for any amount tested except for 20 mg lactose(Table 2). Addition of 10 mM sodium citrate (pH 5.5) and potassiumphosphate (pH 5.5) improved both initial recovered potency and long-termstability of the Clostridial toxin active ingredient in Clostridialtoxin pharmaceutical compositions containing lactose as the singleexcipient. Initial recovered potency increased from about 41% to about60% when the lactose formulation comprised 10 mM sodium citrate (pH 5.5)and increased from about 41% to about 71% when the lactose formulationcomprised 10 mM potassium phosphate (pH 5.5) (Table 2). In addition,increased recovered potency of the Clostridial toxin active ingredientwas also observed after at least one-year of storage using either pH 5.5buffer, as opposed to water, in Clostridial toxin pharmaceuticalcompositions stored at ambient or freezing temperatures (Table 2).However, addition of 10 mM sodium citrate (pH 6.5) to Clostridial toxinpharmaceutical compositions containing lactose as the single excipientdid not improve either initial recovered potency or long-term stabilityof the Clostridial toxin active ingredient (Table 2). Surprisingly,addition of 10 mM potassium phosphate (pH 6.5) to Clostridial toxinpharmaceutical compositions containing lactose as the single excipientactually eliminated recovery of the Clostridial toxin active ingredientaltogether (Table 2). Lastly, the addition of 10 mM sodium chloride toClostridial toxin pharmaceutical compositions containing lactose as thesingle excipient did not improve either recovered potency or long-termstability of the Clostridial toxin active ingredient (Table 2).

The disaccharides sucrose and trehalose and the trisaccharide raffinoseshowed no recovered potency of the Clostridial toxin active ingredientwhatsoever when used as the single excipient. Furthermore, with oneexception, the addition of buffers or sodium chloride to Clostridialtoxin pharmaceutical compositions containing these sugars as the singleexcipient did not improve either initial recovered potency or long-termstability of the Clostridial toxin active ingredient (Table 2). Thesingle exception was the Clostridial toxin pharmaceutical compositioncomprising sucrose and 10 mM sodium citrate (pH 5.5). This formulationexhibited 44% initial recovered potency of the Clostridial toxin activeingredient and this degree of recover was maintained for at least oneyear when stored at either ambient or freezing temperatures (Table 2).

Clostridial toxin pharmaceutical compositions containing a polyol(mannitol) as the single excipient did not exhibit any recovered potency(Table 2). Addition of buffers or sodium chloride to Clostridial toxinpharmaceutical compositions containing mannitol as the single excipientdid not improve either recovered potency or long-term stability of theClostridial toxin active ingredient (Table 2).

When the single excipient used was a non-protein polymer, recoveredpotency of the Clostridial toxin active ingredient dependent on the bothtype of non-protein polymer used and the specific buffer added. Forexample, Dextran 3K and Dextran 40K showed no initial recovered potencyof the Clostridial toxin active ingredient whatsoever when used as thesingle excipient. On the other hand, the addition of about 60 mg of PEG3350 (about 2% (w/v)) resulted in an initial recovered potency of about47%. Similarly the addition of about 5 mg to about 20 mg of PVP 17(about 0.5% (w/v) to about 2% (w/v)) resulted in an initial recoveredpotency of about 39% to about 52% (Table 2).

In general, the addition various buffers did not improve the initialrecovered potency of the Clostridial toxin active ingredient whenDextran 3K or Dextran 40K was used as the single excipient. The soleexception was a Clostridial toxin pharmaceutical compositions comprisingdextran 3K in 10 mM potassium phosphate (pH 5.5), where initialrecovered potency increased from 0% to about 66%, a recovered potencythat was maintained for at least one year. Surprisingly, the additionvarious buffers dramatically improved both recovered potency andlong-term stability of the Clostridial toxin active ingredient when PEG3350 or PVP 17 was used as the single excipient. For example, inClostridial toxin pharmaceutical compositions comprising PEG 3350, theaddition of 10 mM sodium citrate (pH 5.5) increased recovered potencyfrom 0% to about 76%; the addition of 10 mM potassium phosphate (pH 5.5)increased recovered potency from 0% to about 80%; and the addition of 10mM histidine buffer (pH 5.5) increased recovered potency from 0% toabout 72% (Table 2). In all cases, the addition of these various buffersresulted in long-term stability of at least one-year both at ambient andfreezing temperatures.

TABLE 2 Formulations using Botulinum Neurotoxin Complex^(a) - OneExcipient Recovered Potency^(b) (%) Excipient 1 Ambient Below FrerezingAmount Temperatrue^(c) Temperature^(d) Type (mg) Ratio Solution (pH)Initial 3 months 12 months 3 months 12 months Sucrose 5 — Water (pH 5.6)0 0 0 0 0 Sucrose 10 — Water (pH 5.6) 0 0 0 0 0 Sucrose 20 — Water (pH5.6) 0 0 0 0 0 Sucrose 30 — Water (pH 5.3) 0 0 0 0 0 Sucrose 50 — Water(pH 5.6) 0 0 0 0 0 Sucrose 100 — Water (pH 5.6) 0 0 0 0 0 Sucrose 250 —Water (pH 5.6) 0 0 0 0 0 Sucrose 20 — 10 mM SC (pH 5.5) 44 44 44 39 49Sucrose 20 — 10 mM SC (pH 6.5) 0 0 0 0 0 Sucrose 20 — 10 mM PP (pH 5.5)0 0 0 0 0 Sucrose 20 — 10 mM PP (pH 6.5) 0 0 0 0 0 Sucrose 20 — 10 mMNaCl (pH 5.5) 0 0 0 0 0 Sucrose 30 — 10 mM NaCl (pH 5.4) 0 0 0 0 0Sucrose 60 — 10 mM NaCl (pH 5.3) 46 0 0 46 46 Lactose 5 — Water (pH 4.8)0 0 0 0 0 Lactose 10 — Water (pH 4.8) 15 Lactose 20 — Water (pH 4.8) 4145 38 38 51 Lactose 50 — Water (pH 4.8) 35 Lactose 20 — 10 mM SC (pH5.5) 60 55 55 85 67 Lactose 20 — 10 mM SC (pH 6.5) 45 0 0 49 49 Lactose20 — 10 mM PP (pH 5.5) 71 46 49 58 55 Lactose 20 — 10 mM PP (pH 6.5) 0 00 0 0 Lactose 20 — 10 mM NaCl (pH 4.8) 39 50 0 58 — Trehalose 5 — Water0 0 0 0 0 Trehalose 10 — Water 0 0 0 0 0 Trehalose 50 — Water 0 0 0 0 0Raffinose 5 — Water 0 0 0 0 0 Raffinose 10 — Water 0 0 0 0 0 Raffinose50 — Water 0 0 0 0 0 Mannitol 5 — Water 0 0 0 0 0 Mannitol 10 — Water 00 0 0 0 Mannitol 20 — Water 0 0 0 0 0 Mannitol 50 — Water 0 0 0 0 0Mannitol 20 — 10 mM PP (pH 5.5) 0 0 0 0 0 Inulin 5 — Water 0 0 0 0 0Inulin 10 — Water 0 0 0 0 0 Inulin 50 — Water 0 0 0 0 0 Detran 3K 60 —Water (pH 5.2) 0 0 0 0 0 Detran 3K 60 — 10 mM SC (pH 5.5) 0 0 0 0 0Detran 3K 60 — 10 mM SC (pH 6.5) 0 0 0 0 0 Detran 3K 60 — 10 mM PP (pH5.5) 66 0 0 66 73 Detran 3K 60 — 10 mM PP (pH 6.5) 0 0 0 0 0 Detran 3K60 — 10 mM HB (pH 5.5) 0 0 0 0 0 Detran 3K 60 — 10 mM HB (pH 6.5) 0 0 00 0 Detran 40K 60 — Water (pH 5.2) 0 0 0 0 0 Detran 40K 60 — 10 mM SC(pH 5.5) 0 0 0 0 0 Detran 40K 60 — 10 mM SC (pH 6.5) 0 0 0 0 0 Detran40K 60 — 10 mM PP (pH 5.5) 0 0 0 0 0 Detran 40K 60 — 10 mM PP (pH 6.5) 00 0 0 0 Detran 40K 60 — 10 mM HB (pH 5.5) 0 0 0 0 0 Detran 40K 60 — 10mM HB (pH 6.5) 0 0 0 0 0 PVP 17 0.5 — Water (pH 4.2) 0 0 0 0 0 PVP 17 5— Water (pH 4.2) 48 — — — — PVP 17 10 — Water (pH 4.2) 52 — — — — PVP 1720 — Water (pH 4.2) 43 0 0 55 52 PVP 17 30 — Water (pH 4.0) 0 0 0 0 0PVP 17 50 — Water (pH 4.2) 0 0 0 0 0 PVP 17 60 — Water (pH 4.0) 55 0 046 46 PVP 17 100 — Water (pH 4.2) 0 0 0 0 0 PVP 17 250 — Water (pH 4.2)0 0 0 0 0 PVP 17 20 — 10 mM SC (pH 5.5) 113 70 41 101 115 PVP 17 20 — 10mM SC (pH 6.5) 81 44 0 88 58 PVP 17 20 — 10 mM PP (pH 5.5) 79 0 0 75 73PVP 17 20 — 10 mM PP (pH 6.5) 83 0 0 69 69 PVP 17 60 — 10 mM NaCl (pH3.1) 100 0 0 100 100 PVP 17 20 — 10 mM NaCl (pH 4.2) 44 0 0 44 — PVP 1730 — 10 mM NaCl (pH 4.0) 46 — — 58 62 PEG 3350 60 — Water (pH 7.0) 47 00 47 47 PEG 3350 50 — Water (pH 7.0) 0 0 0 0 0 PEG 3350 60 — 10 mM SC(pH 5.5) 76 58 0 87 82 PEG 3350 60 — 10 mM SC (pH 6.5) 57 0 0 57 66 PEG3350 60 — 10 mM PP (pH 5.5) 80 0 0 70 97 PEG 3350 60 — 10 mM PP (pH 6.5)0 0 0 0 0 PEG 3350 60 — 10 mM HB (pH 5.5) 72 97 87 110 74 PEG 3350 60 —10 mM HB (pH 6.5) 73 73 76 59 62 Poloxamer 188 50 — Water 0 0 0 0 0Poloxamer 188 20 — Water (pH 6.5) 0 0 0 0 0 Poloxamer 188 20 — 10 mM SC(pH 5.5) 81 73 67 87 97 Poloxamer 188 20 — 10 mM SC (pH 6.5) 56 0 0 5038 Poloxamer 188 20 — 10 mM PP (pH 5.5) 39 0 0 0 0 Poloxamer 188 20 — 10mM PP (pH 6.5) 0 0 0 0 0 Poloxamer 188 20 — 10 mM NaCl (pH 6.4) 0 0 0 00 Glycine 5 — Water 0 0 0 0 0 Glycine 10 — Water 0 0 0 0 0 Glycine 50 —Water 0 0 0 0 0 ^(a)Amount of botulinum neurotoxin serotype A complexadded per formulation was 150 units. Total volume of formulation was 1.0mL. ^(b)Recovery is expressed as a percentage and is calculated bydividing the potency of the active ingredient determined afterreconstitution divided by the potency of the active ingredientdetermined before addition to the formulation. 3 months refers to thelength of time a formulation was minimally stored at the indicatedtemperature. 12 months refers to the length of time a formulation wasminimally stored at the indicated temperature. ^(c)Ambient temperatureis between about 18° C. to about 22° C. ^(d)Below freezing temperatureis between about −5° C. to about −20° C.

Similar, but more complex results were observed in Clostridial toxinpharmaceutical compositions comprising PVP 17 as the single excipient.For example, in Clostridial toxin pharmaceutical compositions comprisingPVP 17, the addition of 10 mM sodium citrate (pH 5.5) increased initialrecovered potency from about 43% to about 113%; the addition of 10 mMsodium citrate (pH 6.5) increased initial recovered potency from about43% to about 81%; the addition of 10 mM potassium phosphate (pH 5.5)increased initial recovered potency from about 43% to about 97%; and theaddition of 10 mM potassium phosphate (pH 5.5) increased initialrecovered potency from about 43% to about 83%. However, while allbuffers tested exhibited increased recovered potency of the Clostridialtoxin active ingredient, only the addition of the sodium citrate buffersresulted in long-term stability of at least one year. Lastly, theaddition of 10 mM sodium chloride to pharmaceutical compositionscontaining PVP 17 as the single excipient did not improve either initialrecovered potency or long-term stability of the Clostridial toxin activeingredient.

When the single excipient used was a surfactant, recovered potency ofthe Clostridial toxin active ingredient dependent was not detected. Inaddition, use of various buffers resulted in mixed recovered potency.For example, in Clostridial toxin pharmaceutical compositions comprisingPoloxamer 188, the addition of 10 mM sodium citrate (pH 5.5) increasedinitial recovered potency from 0% to about 81%; the addition of 10 mMsodium citrate (pH 6.5) increased initial recovered potency from 0% toabout 56%; and the addition of 10 mM potassium phosphate (pH 5.5)increased initial recovered potency from 0% to about 39%; but theaddition of 10 mM potassium phosphate (pH 6.5) did not improve recoveryat all (Table 2). However, only the addition of 10 mM sodium citrate (pH5.5) resulted in long-term stability of the Clostridial toxin activeingredient stored at either ambient or freezing temperatures (Table 2).

Thus, generally, Clostridial toxin pharmaceutical compositionscomprising a single excipient does not result in significant recoveredpotency of the Clostridial toxin active ingredient, especially when suchcompositions ate stored for at least one year. Surprisingly, however,both the addition of a buffer to the Clostridial toxin pharmaceuticalcomposition can result in both improved recovered potency and increasedlong-term stability of the Clostridial toxin active ingredient. However,the pairing of a particular excipient with a specific buffer can only bedetermined empirically.

Example 2 Non-Protein Stabilized Formulations—Two Excipients

Experiments were carried out to determine the effects of formulationscomprising two different non-protein excipients on Clostridial toxinactive ingredient recovery after reconstitution. The non-proteinexcipients tested were added separately or in combination with thelisted buffers or salts (Tables 3-5). All of the formulations werecompounded, lyophilized, reconstituted and potency assessed in the samemanner, and with the same Clostridial toxin active ingredient used ineach formulation, except that each formulation was prepared withdifferent non-protein excipients or with different amounts of thenon-protein excipients.

The tested formulations were compounded, processed, stored andreconstituted as described in Example 1. Recovered potency wasdetermined using the mouse LD₅₀ bioassay described in Example 1.Recovery is expressed as a percentage and is calculated by dividing thepotency of the Clostridial toxin active ingredient in the storedreconstitution formulation by the potency of the active Clostridialtoxin ingredient determined prior to its addition into the testsolution. The results show that a Clostridial toxin pharmaceuticalcomposition comprising a Clostridial toxin complex could be stabilizedwhen the formulation comprised two non-protein excipients (Tables 3-5).

Clostridial toxin pharmaceutical compositions comprising two differentsugars yielded mixed results. For example, Clostridial toxinpharmaceutical compositions comprising lactose and sucrose did notappear to dramatically improve recovered potency. For example,compositions comprising about 5% (w/v) lactose resulted in an initialrecovered potency of about 35% (Table 2), compositions comprising about5% (w/v) sucrose resulted in no recovered potency (Table 2), andcompositions comprising about 5% (w/v) lactose and about 5% (w/v)sucrose resulted in an initial recovered potency of about 27% (Table 3).Similarly, compositions comprising about 2% (w/v) lactose resulted in aninitial recovered potency of about 41% (Table 2), compositionscomprising about 1% (w/v) sucrose resulted in no recovered potency(Table 2), and compositions comprising about 2% (w/v) lactose and about1% (w/v) sucrose resulted in an initial recovered potency of about 68%(Table 3). Although there was an increased initial recovered potency incompositions comprising both about 2% (w/v) lactose and about 1% (w/v)sucrose, long-term stability of the Clostridial toxin active ingredientin Clostridial toxin pharmaceutical compositions comprising about 2%lactose and about 1% sucrose were similar to compositions comprisingabout 2% (w/v) lactose alone (See Tables 3 & 4).

Similarly, the addition of 10 mM sodium citrate (pH 5.5), 10 mM sodiumcitrate (pH 6.5), and 10 mM potassium phosphate (pH 5.5) had no effecton either initial recovered potency or long-term stability of theClostridial toxin active ingredient in Clostridial toxin pharmaceuticalcompositions comprising about 2% lactose and about 1% sucrose whencompared to compositions comprising about 2% lactose as the sole sugarexcipient. Surprisingly, however, in Clostridial toxin pharmaceuticalcompositions comprising lactose, 2% (w/v), and sucrose, 1% (w/v), theaddition of 10 mM potassium phosphate (pH 6.5) increased initialrecovered potency from 0% to about 50%, and this formulation was stablefor at least one year at freezing temperatures. Similarly striking, inClostridial toxin pharmaceutical compositions comprising lactose, 2%(w/v), and sucrose, 1% (w/v), the addition of 10 mM sodium chlorideincreased initial recovered potency (compare lactose, 2% (w/v), 10 mMsodium chloride at about 39%, sucrose, 2% (w/v), 10 mM sodium chlorideat 0%, and lactose, 2% (w/v), sucrose, 1% (w/v), 10 mM sodium chlorideat about 61%). More importantly, this formulation resulted in long-termstability of at least one year at both ambient and freezingtemperatures.

Clostridial toxin pharmaceutical compositions comprising sucrose andeither trehalose or mannitol did not improve initial recovered potency,with most combinations resulting in no recovery whatsoever. Similarly,Clostridial toxin pharmaceutical compositions comprising lactose andmannitol did not improve initial recovered potency (compare 5% (w/v)lactose at about 35% (Table 2), 5% (w/v) mannitol at 0% (Table 2), and5% (w/v) lactose 5% (w/v) and mannitol at about 23% (Table 3)).

Clostridial toxin pharmaceutical compositions comprising a sugar and anon-protein polymer expanded the range of excipient amounts effective atproducing initial recovered potency and long-term stability of theClostridial toxin active ingredient. For example, various amount ofsucrose in combination with various amount of PVP 17 expanded the rangeof excipient amounts effective at increased recovered potency andlong-term stability of the Clostridial toxin active ingredient. Whensucrose was used as the sole excipient at ranges from about 5 mg toabout 250 mg (about 0.5% (w/v) to about 25% (w/v)), no detectablerecovered potency of a Clostridial toxin active ingredient was observed,whereas PVP 17 at about 5 mg to about 20 mg (about 0.5% (w/v) to about2% (w/v)) resulted in an initial recovered potency. However, Clostridialtoxin pharmaceutical compositions comprising about 30 mg to about 250 mg(about 3% (w/v) to about 25% (w/v)) of sucrose in combination with about30 mg to about 250 mg (about 3% (w/v) to about 25% (w/v)) of PVP 17resulted in about 39 to about 62% initial recovered potency of theClostridial toxin active ingredient (each of these excipients at theseamounts alone resulted in no detectable recovery). As another example,about 5 mg of sucrose (about 0.5% (w/v)) in combination with from about50 mg of PVP 17 (about 5% (w/v)) increased recovered potency of theClostridial toxin active ingredient to about 39% (Table 3) (each ofthese excipients at these amounts alone resulted in no detectablerecovery).

Depending on the amounts added, the addition of various buffers toClostridial toxin pharmaceutical compositions comprising sucrose and PVP17 affected the initial recovered potency or long-term stability of theClostridial toxin active ingredient (Table 3). For example, Clostridialtoxin pharmaceutical compositions comprising about 20 mg sucrose and 10mg PVP 17 resulted in an initial recovered potency of about 77% (Table2). However, the addition of a sodium citrate buffer to this formulationresulted in an increased recovered potency of about 87% to about 100%(Table 3). Furthermore, the addition of an about pH 5.5 sodium citratebuffer to Clostridial toxin pharmaceutical compositions comprising about20 mg sucrose and 10 mg PVP 17 resulted in at least one year long-termstability when stored at either ambient or below freezing temperatures(Table 3). Likewise, although not increasing the degree of initialrecovered potency observed, Clostridial toxin pharmaceuticalcompositions comprising about 20 mg sucrose and 10 mg PVP 17 in about pH5.5 to about pH 6.5 potassium phosphate buffer resulted in significantlyincreased long term stability of the formulations stored at ambienttemperatures (Table 3).

In Clostridial toxin pharmaceutical compositions comprising sucrose andPVP 17, the addition of sodium chloride to the formulation did notappear to have a great effect on initial recovered potency. However,Clostridial toxin pharmaceutical compositions comprising about 20 mgsucrose and 10 mg PVP 17 in sodium chloride resulted in significantlyincreased long term stability of the formulations stored at ambienttemperatures (Table 3).

TABLE 3 Formulations using Botulinum Neurotoxin Complex^(a) - TwoExcipients with One Being a Sugar Recovered Potency^(b) (%) Excipient 1Excipient 2 Ambient Below Frerezing Amount Amount Temperatrue^(c)Temperature^(d) Type (mg) Type (mg) Ratio Solution (pH) Initial 3 months12 months 3 months 12 months Sucrose 50 Lactose 50 1:1 Water (pH 4.8) 27Sucrose 10 Lactose 20 1:2 Water (pH 4.8) 68 44  44  46 50 Sucrose 10Lactose 20 1:2 10 mM SC (pH 5.5) 64 68  58  65 65 Sucrose 10 Lactose 201:2 10 mM SC (pH 6.5) 41 0 0 41 0 Sucrose 10 Lactose 20 1:2 10 mM PP (pH5.5) 43 55  49  55 68 Sucrose 10 Lactose 20 1:2 10 mM PP (pH 6.5) 50 0 051 38 Sucrose 10 Lactose 20 1:2 10 mM NaCl (pH 4.8) 61 58  45  66 58Sucrose 50 Trehalose 50 1:1 Water (pH 4.3) 0 0 0 0 0 Sucrose 50Trehalose 5 10:1  Water (pH 4.3) 0 0 0 0 0 Sucrose 5 Trehalose 50  1:10Water (pH 4.3) 0 0 0 0 0 Sucrose 50 Mannitol 5 10:1  Water (pH 4.3) 0 00 0 0 Sucrose 50 Mannitol 50 1:1 Water (pH 4.3) 27 — — — — Sucrose 5Mannitol 50  1:10 Water (pH 4.3) 0 0 0 0 0 Sucrose 250 PVP 17 10 25:1 Water (pH 4.3) 58 — — — — Sucrose 5 PVP 17 0.5 10:1  Water (pH 4.3) 0 00 0 0 Sucrose 20 PVP 17 10 2:1 Water (pH 4.3) 77 49  0 76 101 Sucrose250 PVP 17 250 1:1 Water (pH 4.3) 39 — — — — Sucrose 30 PVP 17 30 1:1Water (pH 4.1) 62 — — — — Sucrose 15 PVP 17 15 1:1 Water (pH 4.1) 77 0 068 80 Sucrose 5 PVP 17 5 1:1 Water (pH 4.3) 49 — — — — Sucrose 0.5 PVP17 0.5 1:1 Water (pH 4.3) 0 0 0 0 0 Sucrose 5 PVP 17 10 1:2 Water (pH4.3) 58 — — — — Sucrose 5 PVP 17 20 1:4 Water (pH 4.3) 47 — — — —Sucrose 5 PVP 17 50  1:10 Water (pH 4.3) 39 — — — — Sucrose 0.5 PVP 17 5 1:10 Water (pH 4.3) 58 — — — — Sucrose 5 PVP 17 100  1:20 Water (pH4.3) 0 0 0 0 0 Sucrose 0.5 PVP 17 10  1:20 Water (pH 4.3) 46 — — — —Sucrose 0.5 PVP 17 20  1:40 Water (pH 4.3) 49 — — — — Sucrose 20 PVP 1710 2:1 10 mM SC (pH 5.5) 100 52  38  87 101 Sucrose 20 PVP 17 10 2:1 10mM SC (pH 6.5) 87 0 0 88 85 Sucrose 20 PVP 17 10 2:1 10 mM PP (pH 5.5)65 42  47  83 87 Sucrose 20 PVP 17 10 2:1 10 mM PP (pH 6.5) 63 61  51 75 99 Sucrose 20 PVP 17 10 2:1 10 mM NaCl (pH 4.4) 83 112  43  77 93Sucrose 30 PVP 17 30 1:1 10 mM NaCl (pH 4.1) 66 0 0 66 66 Sucrose 15 PVP17 15 1:1 10 mM NaCl (pH 4.1) 59 0 0 59 59 Sucrose 50 PEG 3350 5 10:1 Water 41 — — — — Sucrose 50 PEG 3350 50 1:1 Water 44 — — — — Sucrose 5PEG 3350 50  1:10 Water 35 — — — — Sucrose 10 Poloxamer 188 0.25 40:1 Water (pH 5.9) 62 0 0 78 78 Sucrose 5 Poloxamer 188 0.125 40:1  Water(pH 5.7) 70 0 0 70 78 Sucrose 60 Poloxamer 188 3 20:1  Water (pH 6.1) 750 0 84 106 Sucrose 30 Poloxamer 188 1.5 20:1  Water (pH 6.1) 104 0 0 9279 Sucrose 10 Poloxamer 188 0.5 20:1  Water (pH 6.2) 66 0 0 88 79Sucrose 5 Poloxamer 188 0.25 20:1  Water (pH 5.9) 64 0 0 78 78 Sucrose55 Poloxamer 188 5.5 10:1  Water (pH 6.7) 99 0 0 115 115 Sucrose 50Poloxamer 188 5 10:1  Water 43 — — — — Sucrose 27 Poloxamer 188 2.710:1  Water (pH 6.2) 92 0 0 80 80 Sucrose 48 Poloxamer 188 12 4:1 Water(pH 6.4) 110 0 0 85 113 Sucrose 24 Poloxamer 188 6 4:1 Water (pH 6.4)102 0 0 88 84 Sucrose 10 Poloxamer 188 2.5 4:1 Water (pH 6.4) 84 0 0 10487 Sucrose 5 Poloxamer 188 1.25 4:1 Water (pH 6.4) 72 0 0 92 82 Sucrose40 Poloxamer 188 20 2:1 Water (pH 6.9) 113 78  74  102 111 Sucrose 20Poloxamer 188 10 2:1 Water (pH 6.5) 101 87  0 117 115 Sucrose 10Poloxamer 188 5 2:1 Water (pH 6.9) 94 0 0 92 106 Sucrose 5 Poloxamer 1882.5 2:1 Water (pH 6.6) 105 61  0 108 102 Sucrose 2.5 Poloxamer 188 1.252:1 Water (pH 6.4) 87 — — 85 86 Sucrose 1.25 Poloxamer 188 0.625 2:1Water (pH 6.2) 71 — — 81 90 Sucrose 50 Poloxamer 188 50 1:1 Water 59 — —— — Sucrose 20 Poloxamer 188 40 1:2 Water (pH 6.9) 115 117  101  117 115Sucrose 5 Poloxamer 188 50  1:10 Water 55 — — — — Sucrose 60 Poloxamer188 6 10:1  10 mM SC (pH 5.5) 111 97  101 115 Sucrose 40 Poloxamer 18820 2:1 10 mM SC (pH 5.5) 113 112  89  115 101 Sucrose 20 Poloxamer 18810 2:1 10 mM SC (pH 5.5) 77 81  101  81 115 Sucrose 5 Poloxamer 188 2.52:1 10 mM SC (pH 5.5) 92 — — 90 102 Sucrose 2.5 Poloxamer 188 1.25 2:110 mM SC (pH 5.5) 80 — — 102 80 Sucrose 1.25 Poloxamer 188 0.625 2:1 10mM SC (pH 5.5) 106 — — 102 77 Sucrose 0.625 Poloxamer 188 0.3125 2:1 10mM SC (pH 5.5) 80 — — 92 92 Sucrose 20 Poloxamer 188 10 2:1 10 mM SC (pH6.5) 90 91  67  115 97 Sucrose 20 Poloxamer 188 10 2:1 10 mM PP (pH 5.5)112 113  0 113 115 Sucrose 20 Poloxamer 188 10 2:1 10 mM PP (pH 6.5) 9390  63  119 84 Sucrose 20 Poloxamer 188 40 1:2 10 mM SC (pH5.5) 107 115 101  115 117 Sucrose 30 Poloxamer 188 1.5 20:1  10 mM NaCl (pH 6.0) 1040 0 104 102 Sucrose 55 Poloxamer 188 5.5 10:1  10 mM NaCl (pH 6.1) 104 00 84 84 Sucrose 27 Poloxamer 188 2.7 10:1  10 mM NaCl (pH 6.1) 102 0 096 81 Sucrose 48 Poloxamer 188 12 4:1 10 mM NaCl (pH 6.2) 96 0 0 97 92Sucrose 24 Poloxamer 188 6 4:1 10 mM NaCl (pH 6.2) 100 0 0 66 0 Sucrose40 Poloxamer 188 20 2:1 10 mM NaCl (pH 6.4) 84 80  80  102 102 Sucrose20 Poloxamer 188 10 2:1 10 mM NaCl (pH 6.2) 117 50  89  115 117 Sucrose5 Poloxamer 188 2.5 2:1 10 mM NaCl (pH 6.1) 87 — — 106 92 Sucrose 2.5Poloxamer 188 1.25 2:1 10 mM NaCl (pH 6.0) 92 — — 82 102 Sucrose 1.25Poloxamer 188 0.625 2:1 10 mM NaCl (pH 5.8) 85 — — 92 105 Sucrose 0.625Poloxamer 188 0.3125 2:1 10 mM NaCl (pH 5.8) 92 — — 78 92 Sucrose 10Polysorbate 80 0.5 20:1  Water (pH 5.8) 98 — — 82 82 Sucrose 5Polysorbate 80 0.25 20:1  Water (pH 5.8) 78 — — 92 92 Sucrose 10Polysorbate 80 2.5 4:1 Water (pH 6.0) 96 — — 104 104 Sucrose 5Polysorbate 80 1.25 4:1 Water (pH 6.0) 102 — — 90 90 Sucrose 50 Glycine50 1:1 Water 0 0 0 0 0 Sucrose 50 Glycine 5 10:1  Water 0 0 0 0 0Sucrose 5 Glycine 50  1:10 Water 0 0 0 0 0 Lactose 50 Mannitol 50 1:1Water 23 — — — — Lactose 5 PVP 17 0.5 10:1  Water 52 — — — — Lactose 5PVP 17 5 1:1 Water 57 — — — — Lactose 0.5 PVP 17 0.5 1:1 Water 0 0 0 0 0Lactose 5 PVP 17 10 1:2 Water 65 — — — — Lactose 5 PVP 17 20 1:4 Water49 — — — — Lactose 5 PVP 17 50  1:10 Water 52 — — — — Lactose 0.5 PVP 175  1:10 Water 65 — — — — Lactose 5 PVP 17 100  1:20 Water 0 — — — —Lactose 0.5 PVP 17 10  1:20 Water 47 — — — — Lactose 0.5 PVP 17 20  1:40Water 65 — — — — Lactose 0.5 PVP 17 50  1:100 Water 0 — — — — Lactose 55PEG 3550 5.5 10:1  Water (pH 4.9) 96 61  62  112 98 Lactose 40 PEG 355020 2:1 Water (pH 5.6) 96 58  55  110 117 Lactose 50 PEG 3350 50 1:1Water 53 — — — — Lactose 55 PEG 3550 5.5 10:1  10 mM SC (pH 5.5) 96 62 62  82 102 Lactose 40 PEG 3550 20 2:1 10 mM SC (pH 5.5) 79 66  70  92104 Lactose 55 Poloxamer 188 5.5 10:1  Water (pH 4.7) 108 80  55  106 92Lactose 40 Poloxamer 188 20 2:1 Water (pH 5.9) 88 60  46  107 104Lactose 20 Poloxamer 188 10 2:1 Water (pH 5.6) 63 69  0 95 113 Lactose 5Poloxamer 188 2.5 2:1 Water (pH 5.8) 107 — — 110 106 Lactose 2.5Poloxamer 188 1.25 2:1 Water (pH 5.8) 87 — — 92 82 Lactose 1.25Poloxamer 188 0.625 2:1 Water (pH 5.7) 92 — — 96 104 Lactose 0.625Poloxamer 188 0.3125 2:1 Water (pH 5.6) 73 — — 66 100 Lactose 55Poloxamer 188 5.5 10:1  10 mM SC (pH 5.5) 100 78  58  96 102 Lactose 40Poloxamer 188 20 2:1 10 mM SC (pH 5.5) 93 66  59  107 122 Lactose 20Poloxamer 188 10 2:1 10 mM SC (pH 5.5) 101 73  69  99 117 Lactose 5Poloxamer 188 2.5 2:1 10 mM SC (pH 5.5) 107 — — 92 112 Lactose 2.5Poloxamer 188 1.25 2:1 10 mM SC (pH 5.5) 86 — — 102 101 Lactose 1.25Poloxamer 188 0.625 2:1 10 mM SC (pH 5.5) 106 — — 90 107 Lactose 0.625Poloxamer 188 0.3125 2:1 10 mM SC (pH 5.5) 74 — — 61 81 Lactose 20Poloxamer 188 10 2:1 10 mM SC (pH 6.5) 81 56  56  113 117 Lactose 20Poloxamer 188 10 2:1 10 mM PP (pH 5.5) 115 88  85  107 114 Lactose 20Poloxamer 188 10 2:1 10 mM PP (pH 6.5) 91 65  65  103 93 Lactose 20Poloxamer 188 10 2:1 10 mM NaCl (pH 5.5) 115 87  101  115 115 Lactose 5Poloxamer 188 2.5 2:1 10 mM NaCl (pH 5.5) 107 — — 92 112 Lactose 2.5Poloxamer 188 1.25 2:1 10 mM NaCl (pH 5.5) 86 — — 102 101 Lactose 1.25Poloxamer 188 0.625 2:1 10 mM NaCl (pH 5.5) 106 — — 90 107 Lactose 0.625Poloxamer 188 0.3125 2:1 10 mM NaCl (pH 5.5) 74 — — 61 81 Trehalose 50Mannitol 50 10:1  Water 0 0 0 0 0 Trehalose 50 Mannitol 5 1:1 Water 0 00 0 0 Trehalose 5 Mannitol 50  1:10 Water 0 0 0 0 0 Trehalose 50 PEG3350 5 10:1  Water 41 — — — — Trehalose 50 PEG 3350 50 1:1 Water 0 0 0 00 Trehalose 5 PEG 3350 50  1:10 Water 36 — — — — Trehalose 50 Poloxamer188 5 10:1  Water 50 — — — — Trehalose 50 Poloxamer 188 50 1:1 Water 53— — — — Trehalose 5 Poloxamer 188 50  1:10 Water 75 — — — — Trehalose 50Glycine 5 10:1  Water 0 0 0 0 0 Trehalose 50 Glycine 50 1:1 Water 0 0 00 0 Trehalose 5 Glycine 50  1:10 Water 0 0 0 0 0 ^(a)Amount of botulinumneurotoxin serotype A complex added per formulation was 150 units. Totalvolume of formulation was 1.0 mL. ^(b)Recovery is expressed as apercentage and is calculated by dividing the potency of the activeingredient determined after reconstitution divided by the potency of theactive ingredient determined before addition to the formulation. 3months refers to the length of time a formulation was minimally storedat the indicated temperature. 12 months refers to the length of time aformulation was minimally stored at the indicated temperature.^(c)Ambient temperature is between about 18° C. to about 22° C.^(d)Below freezing temperature is between about −5° C. to about −20° C.

As another example, although no detectable recovered potency wasobserved when about 5 mg to about 50 mg of sucrose (about 0.5 (w/v) toabout 5% (w/v)) was used as the sole excipient, or when about 50 mg ofPEG 3350 (about 5% (w/v)) was used as the sole excipient, in combinationabout 35% to about 44% increased recovered potency of the Clostridialtoxin active ingredient was exhibited (Table 3).

Clostridial toxin pharmaceutical compositions comprising lactose and anon-protein polymer also expanded the range of excipient amountseffective at producing initial recovered potency and long-term stabilityof the Clostridial toxin active ingredient. For example, when used asthe sole excipient, lactose was effective at increasing recoveredpotency at about 10 mg to about 50 mg (about 1% (w/v) to about 5% (w/v))(Table 2), whereas PVP 17 was effective at increasing recovered potencyat about 5 mg to about 20 mg (about 0.5% (w/v) to about 2% (w/v)) (Table2). However, about 5 mg of lactose (about 0.5% (w/v)) in combinationwith from about 0.5 mg of PVP 17 (about 0.05% (w/v)) increased initialrecovered potency of the Clostridial toxin active ingredient to about52% (Table 3) (each of these excipients at these amounts alone resultedin no detectable recovery, see Table 2). As another example, about 5 mgof lactose (about 0.5% (w/v)) in combination with from about 50 mg ofPVP 17 (about 5% (w/v)) increased initial recovered potency of theClostridial toxin active ingredient to about 52% (Table 3) (each ofthese excipients at these concentrations alone resulted in no detectablerecovery, see Table 2).

Furthermore, the addition of lactose, at amounts this sugar alone isineffective to produce initial recovered potency of the Clostridialtoxin active ingredient, appeared to enhance initial recovered potencyin Clostridial toxin pharmaceutical compositions comprising an amount ofPVP 17 sufficient to produce an initial recovered potency as the soleexcipient. For example, about 5 mg of lactose (0.5% (w/v)) incombination with about 5 mg to about 20 mg of PVP 17 (about 0.5% (w/v)to about 2% (w/v)) increased initial recovered potency of theClostridial toxin active ingredient to about 57%, about 65%, and about49%, respectively (Table 3). This recovered potency is significantlyhigher that the recovery observed when PVP 17 is used as the soleexcipient (See Table 2, 5 mg of PVP 17, 0.5% (w/v) alone at about 48%;10 mg of PVP 17, 1% (w/v) alone at about 52%; 20 mg of PVP 17, 2% (w/v)alone at about 43%). Similarly, about 0.5 mg of lactose (0.05% (w/v)) incombination with about 5 mg to about 20 mg of PVP 17 (about 0.5% (w/v)to about 2% (w/v)) increased initial recovered potency of theClostridial toxin active ingredient to about 65%, about 47%, and about65%, respectively (Table 3). In general, this recovered potency issignificantly higher that the recovery observed when PVP 17 is used asthe sole excipient (See Table 2, 5 mg of PVP 17, 0.5% (w/v) alone atabout 48%; 10 mg of PVP 17, 1% (w/v) alone at about 52%; 20 mg of PVP17, 2% (w/v) alone at about 43%).

Similar results were seen when lactose was combined with PEG 3350.Clostridial toxin pharmaceutical compositions comprising about 50 mglactose (about 5% (w/v)) resulted in an initial recovered potency of 35%(Table 2), whereas, Clostridial toxin compositions comprising about 50mg PEG 3350 (about 5% (w/v)) resulted in no initial recovered potency ofthe Clostridial toxin active ingredient (Table 2). However, Clostridialtoxin compositions comprising about 50 mg lactose (about 5% (w/v)) andabout 50 mg PEG 3350 (about 5% (w/v)) resulted in an initial recoveredpotency of 53% (Table 3). Enhancement of initial recovered potency wasalso observed in Clostridial toxin compositions comprising about lactoseand PEG 3350 in various buffered solutions (see Table 3).

Clostridial toxin pharmaceutical compositions comprising a sugar and asurfactant resulted in an effective increased recovered potency andlong-term stability of the Clostridial toxin active ingredient over awide range of excipient amounts. For example, both sucrose alone andPoloxamer 188 alone resulted in no detectable recovered potency of aClostridial toxin active ingredient (Table 2). Surprisingly Clostridialtoxin pharmaceutical compositions comprising from about 1.25 mg to about60 mg of sucrose (about 0.125% (w/v) to about 6% (w/v)) in combinationwith about 0.25 mg to about 50 mg of Poloxamer 188 (about 0.025% (w/v)to about 5% (w/v)), all resulted in increased recovered potency of theClostridial toxin active ingredient of about 43% to about 115% (Table3). In addition, all such combinations resulted in long-term stabilityof at least one year of the Clostridial toxin active ingredient whenstored at least at below freezing temperatures (Table 3).

Interestingly, in Clostridial toxin pharmaceutical compositionscomprising sucrose and Poloxamer 188, the addition of various buffers tothe formulation did not appear to have a great effect on initialrecovered potency or long-term stability of the Clostridial toxin activeingredient when stored at below freezing temperatures (Table 3).Surprisingly, however, the addition of various buffers to Clostridialtoxin pharmaceutical compositions comprising sucrose and Poloxamer 188dramatically improved long-term stability of the Clostridial toxinactive ingredient when stored at ambient temperatures (Table 3). Theaddition of sodium chloride to Clostridial toxin pharmaceuticalcompositions comprising sucrose and Poloxamer 188 did not appear to havea great affect on initial recovered potency or long-term stability ofthe Clostridial toxin active ingredient (Table 3).

Similar results where seen when sucrose was combined with polysorbate80. Clostridial toxin compositions comprising about sucrose as the soleexcipient resulted in no detectable recovered potency of a Clostridialtoxin active ingredient (Table 2). However, Clostridial toxincompositions comprising about 10 mg to about 20 mg sucrose (about 1%(w/v) to about 2% (w/v)) and about 0.25 mg to about 2.5 mg polysorbate80 (about 0.025% (w/v) to about 0.25% (w/v)) resulted in an initialrecovered potency of about 78% to about 102% (Table 3). The enhancementof long term stability was also observed in Clostridial toxincompositions comprising about sucrose and polysorbate 80 (see Table 3).

As another example, both sucrose alone and Poloxamer 188 alone resultedin no detectable recovered potency of a Clostridial toxin activeingredient (Table 2). Surprisingly Clostridial toxin pharmaceuticalcompositions comprising from about 1.25 mg to about 60 mg of sucrose(about 0.125% (w/v) to about 6% (w/v)) in combination with about 0.25 mgto about 50 mg of Poloxamer 188 (about 0.025% (w/v) to about 5% (w/v)),all resulted in increased recovered potency of the Clostridial toxinactive ingredient of about 43% to about 115% (Table 3). In addition, allsuch combinations resulted in long-term stability of at least one yearof the Clostridial toxin active ingredient when stored at least at belowfreezing temperatures (Table 3).

Clostridial toxin pharmaceutical compositions comprising lactose andPoloxamer 188 also expanded the range of excipient amounts effective atproducing initial recovered potency and long-term stability of theClostridial toxin active ingredient. For example, when used as the soleexcipient, lactose was effective at recovering the Clostridial toxinactive ingredient at about 10 mg to about 50 mg (about 1% (w/v) to about5% (w/v)) (Table 2), whereas Poloxamer 188 alone resulted in nodetectable recovered potency of a Clostridial toxin active ingredient(Table 2). However, about 0.625 to about 5 mg of lactose (about 0.0625%(w/v) to about 0.5% (w/v)) in combination with about 0.3125 mg to about2.5 mg Poloxamer 188 (about 0.03125% (w/v) to about 0.25% (w/v))increased initial recovered potency of the Clostridial toxin activeingredient to about 73% to about 107 (Table 3) (each of these excipientsat these amounts alone resulted in no detectable recovery, see Table 2).In addition, all such combinations resulted in long-term stability of atleast one year of the Clostridial toxin active ingredient when stored atleast at below freezing temperatures (Table 3).

Furthermore, the addition of Poloxamer 188, at amounts this surfactantalone is ineffective to produce recovery of the Clostridial toxin activeingredient, appeared to enhance initial recovered potency in Clostridialtoxin pharmaceutical compositions comprising an amount of lactosesufficient to produce an initial recovered potency as the soleexcipient. For example, about 20 mg to about 55 mg of lactose (about 2%(w/v) to about 5.5% (w/v)) in combination with about 5.5 mg to about 20mg of Poloxamer 188 (about 0.55% (w/v) to about 2% (w/v)) increasedinitial recovered potency of the Clostridial toxin active ingredient toabout 63% to about 108% (Table 3). This recovered potency issignificantly higher that the recovery observed when lactose was used asthe sole excipient (See Table 2, 10 mg of lactose, 1% (w/v) alone atabout 15%; 20 mg of lactose, 2% (w/v) alone at about 41%; 50 mg oflactose, 5% (w/v) alone at about 35%).

Depending on the amounts added, the addition of various buffers toClostridial toxin pharmaceutical compositions comprising lactose andPoloxamer 188 affected the initial recovered potency or long-termstability of the Clostridial toxin active ingredient (Table 3). Forexample, Clostridial toxin pharmaceutical compositions comprising about20 mg lactose and 10 mg Poloxamer 188 resulted in an initial recoveredpotency of about 63% (Table 2). However, the addition of an about pH 5.5to an about pH 6.5 buffered solution to this formulation resulted in anincreased initial recovered potency of about 81% to about 115% (Table3). Likewise, the addition of a buffer to these formulations resulted inenhanced long-term stability of at least one year when stored at eitherambient or below freezing temperatures. Similarly, the addition ofsodium chloride to Clostridial toxin pharmaceutical compositionscomprising lactose and Poloxamer 188, although not having a dramaticaffect on initial recovered potency, greatly increased long-termstability of the Clostridial toxin active ingredient, especially at whenstored at ambient temperatures (Table 3).

Clostridial toxin pharmaceutical compositions comprising two non-proteinpolymers resulted in enhanced recovered potency and long-term stabilityof the Clostridial toxin active ingredient. For example, the addition ofDextran 3K, at amounts this non-protein polymer alone is ineffective toproduce initial recovered potency of the Clostridial toxin activeingredient, appeared to enhance initial recovered potency in Clostridialtoxin pharmaceutical compositions comprising an amount of PEG 3350sufficient to produce an initial recovered potency as the soleexcipient. Thus, compositions comprising both Dextran 3K and PEG 3350exhibited enhanced initial recovered potency in water (compare 0%initial recovered potency of PEG 3350 alone (Table 2) with 47% initialrecovered potency Dextran 3K and PEG 3350 together (Table 4)); in sodiumcitrate buffers (compare 76% initial recovered potency of PEG 3350 alonein sodium citrate buffer (pH 5.5) (Table 2) with 92% initial recoveredpotency Dextran 3K and PEG 3350 together in sodium citrate buffer (pH5.5) (Table 4); and 57% initial recovered potency of PEG 3350 alone insodium citrate buffer (pH 6.5) (Table 2) with 82% initial recoveredpotency Dextran 3K and PEG 3350 together in sodium citrate buffer (pH6.5) (Table 4)); potassium phosphate buffers (compare 80% initialrecovered potency of PEG 3350 alone in potassium phosphate buffer (pH5.5) (Table 2) with 101% initial recovered potency Dextran 3K and PEG3350 together in potassium phosphate buffer (pH 5.5) (Table 4); and 0%initial recovered potency of PEG 3350 alone in potassium phosphatebuffer (pH 6.5) (Table 2) with 102% initial recovered potency Dextran 3Kand PEG 3350 together in potassium phosphate buffer (pH 5.5) (Table 4));and histidine buffers (compare 72% initial recovered potency of PEG 3350alone in potassium phosphate buffer (pH 5.5) (Table 2) with 82% initialrecovered potency Dextran 3K and PEG 3350 together in histidine buffer(pH 5.5) (Table 4)).

Clostridial toxin pharmaceutical compositions comprising PVP 17 and PEG3350 expanded the range of excipient amounts effective at producinginitial recovered potency and long-term stability of the Clostridialtoxin active ingredient. For example, when PVP 17 was used as the soleexcipient at ranges from about 30 mg to about 250 mg (about 3% (w/v) toabout 25% (w/v)), no detectable recovered potency of a Clostridial toxinactive ingredient was observed, whereas PEG 3350 only resulted ininitial recovered potency at amounts above about 60 mg (about 6% (w/v))(Table 2). However, Clostridial toxin pharmaceutical compositionscomprising about 30 mg to about 40 mg PVP 17 (about 3% (w/v) to about 4%(w/v)) in combination with about 20 mg to about 30 mg of PEG 3350 (about2% (w/v) to about 3% (w/v)) resulted in initial recovered potency ofabout 80% (Table 4) (each of these excipients alone resulted in nodetectable initial recovered potency). Likewise, when PEG 3350 was usedas the sole excipient at ranges above about 60 mg (about 6% (w/v)), nodetectable recovered potency of a Clostridial toxin active ingredientwas observed, whereas PVP 17 at about 5 mg to about 20 mg (about 0.5%(w/v) to about 2% (w/v)) resulted in an initial recovered potency (Table2). However, Clostridial toxin pharmaceutical compositions comprisingabout 40 mg to about 55 mg (about 4% (w/v) to about 5.5% (w/v)) of PEG3350 in combination with about 20 mg (about 2% (w/v)) of PVP 17 resultedin about 68% initial recovered potency of the Clostridial toxin activeingredient (20 mg (about 2% (w/v)) of PVP 17 alone resulted in a 43%initial recovered potency) (Table 4). This enhanced initial recovery wasalso observed when various buffered solutions were added to theformulations (Table 4).

Clostridial toxin pharmaceutical compositions comprising a non-proteinpolymer and a surfactant resulted in an effective increased recoveredpotency and long-term stability of the Clostridial toxin activeingredient. For example, both Dextran 3K and Poloxamer 188 aloneresulted in no detectable recovered potency of a Clostridial toxinactive ingredient (Table 2). Surprisingly Clostridial toxinpharmaceutical compositions comprising both Dextran 3K and Poloxamer 188resulted in an initial recovered potency of the Clostridial toxin activeingredient of about 78% to about 98% (Table 4). Furthermore, thissynergistic effect was also observed in Clostridial toxin pharmaceuticalcompositions comprising Dextran 3K and Poloxamer 188 in bufferedsolutions (Table 4). Both Dextran 3K and Poloxamer 188 alone resulted inno detectable recovered potency of a Clostridial toxin active ingredientin formulations comprising sodium citrate buffers or potassium phosphatebuffer (pH 6.5) (Table 2). However, Clostridial toxin pharmaceuticalcompositions comprising Dextran 3K and Poloxamer 188 resulted in about82% to about 100% initial recovered potency with the addition of sodiumcitrate buffer (pH 5.5); about 85% to about 99% initial recoveredpotency with the addition of sodium citrate buffer (pH 6.5); about 82%to about 103% initial recovered potency with the addition of potassiumphosphate buffer (pH 6.5); about 103% to about 125% initial recoveredpotency with the addition of histidine buffer (pH 5.5); and about 115%to about 134% initial recovered potency with the addition of histidinebuffer (pH 6.5). In addition, such buffered Clostridial toxinpharmaceutical compositions resulted in enhanced long-term stability forat least one year. Similarly, enhanced recover was seen in Clostridialtoxin pharmaceutical compositions comprising Dextran 3K and Poloxamer188 in potassium phosphate buffer (pH 5.5) (compare 66% initialrecovered potency for Dextran 3K alone (Table 2); 39% initial recoveredpotency for Poloxamer 188 alone (Table 2); and about 90% to about 120%initial recovered potency for Dextran 3K and Poloxamer 188 together(Table 4). Clostridial toxin pharmaceutical compositions comprisingDextran 3K and Poloxamer 188 in potassium phosphate buffer (pH 5.5) alsodemonstrated enhanced long-term stability when stored at either ambientor below freezing temperatures.

Similar degrees of improved initial recovery and long-term stability wasobserved in Clostridial toxin pharmaceutical compositions comprisingDextran 40K and Poloxamer 188. For example, both Dextran 40K andPoloxamer 188 alone resulted in no detectable recovered potency of aClostridial toxin active ingredient (Table 2). Surprisingly Clostridialtoxin pharmaceutical compositions comprising both Dextran 40K andPoloxamer 188 resulted in an initial recovered potency of theClostridial toxin active ingredient of about 85% to about 102% (Table4). This synergistic effect was also observed in Clostridial toxinpharmaceutical compositions comprising Dextran 40K and Poloxamer 188 inbuffered solutions. Both Dextran 40K and Poloxamer 188 alone resulted inno detectable recovered potency of a Clostridial toxin active ingredientin formulations comprising potassium phosphate buffer (pH 6.5) (Table2). However, Clostridial toxin pharmaceutical compositions comprisingDextran 40K and Poloxamer 188 resulted in about 102% to about 115%initial recovered potency with the addition of potassium phosphatebuffer (pH 6.5) (Table 4). Furthermore, Clostridial toxin pharmaceuticalcompositions comprising Dextran 40K and Poloxamer 188 in various otherbuffered solutions resulted in enhanced recovered potency and long-termstability of the Clostridial toxin active ingredient. Thus, compositionscomprising both Dextran 40K and Poloxamer 188 exhibited enhanced initialrecovered potency in sodium citrate buffers (compare 81% initialrecovered potency of Poloxamer 188 alone in sodium citrate buffer (pH5.5) (Table 2) with 128% initial recovered potency Dextran 40K andPoloxamer 188 together in sodium citrate buffer (pH 5.5) (Table 4); and56% initial recovered potency of Poloxamer 188 alone in sodium citratebuffer (pH 5.5) (Table 2) with 100% initial recovered potency Dextran40K and Poloxamer 188 together in sodium citrate buffer (pH 6.5) (Table4)); and potassium phosphate buffer (pH 5.5) (compare 39% initialrecovered potency of Poloxamer 188 alone in potassium phosphate buffer(pH 5.5) (Table 2) with 103% initial recovered potency Dextran 40K andPoloxamer 188 together in potassium phosphate buffer (pH 5.5) (Table4)).

Clostridial toxin pharmaceutical compositions comprising PVP 17 and asurfactant resulted in an effective increased recovered potency andlong-term stability of the Clostridial toxin active ingredient over awide range of excipient amounts. For example, when used as the soleexcipient, PVP 17 was effective at increasing recovered potency atamounts ranging from about 5 mg to about 20 mg (about 0.5% (w/v) toabout 2% (w/v)). As discussed above, Poloxamer 188 alone resulted in nodetectable recovered potency of a Clostridial toxin active ingredient.However, about 0.3125 mg to about 2.5 mg of PVP 17 (about 0.03% (w/v) toabout 0.25% (w/v)) in combination with from about 0.625 mg to about 5 mgof Poloxamer 188 (about 0.06% (w/v) to about 0.5% (w/v)) increasedrecovered potency of the Clostridial toxin active ingredient to about64% to about 80% (each of these excipients at these concentrations aloneresulted in no detectable recovery). Similarly, about 30 mg to about 60mg of PVP 17 (about 3% (w/v) to about 6% (w/v)) in combination with fromabout 1.5 mg to about 5 mg of Poloxamer 188 (about 0.15% (w/v) to about0.5% (w/v)) increased recovered potency of the Clostridial toxin activeingredient to about 68% to about 77% (each of these excipients at theseconcentrations alone resulted in no detectable recovery). The additionof various buffers or sodium chloride to Clostridial toxinpharmaceutical compositions comprising PVP 17 and Poloxamer 188 did notappear to have a great affect on initial recovered potency or long-termstability of the Clostridial toxin active ingredient (Table 4).

TABLE 4 Formulations using Botulinum Neurotoxin Complex^(a) - TwoExcipients with One Being a Non-Protein Polymer Recovered Potency^(b)(%) Excipient 1 Excipient 2 Ambient Below Frerezing Amount AmountTemperatrue^(c) Temperature^(d) Type (mg) Type (mg) Ratio Solution (pH)Initial 3 months 12 months 3 months 12 months Dextran 3K 55 PEG 3350 5.510:1  Water (pH 7.1) 0 0 0 0 0 Dextran 3K 40 PEG 3350 20 2:1 Water (pH6.3) 82 0 0 0 0 Dextran 3K 30 PEG 3350 30 1:1 Water (pH 6.4) 0 0 0 0 0Dextran 3K 20 PEG 3350 40 1:2 Water (pH 6.7) 47 0 0 0 0 Dextran 3K 5.5PEG 3350 55  1:10 Water (pH 6.9) 47 0 0 0 0 Dextran 3K 55 PEG 3350 5.510:1  10 mM SC (pH 5.5) 82 0 0 92 94 Dextran 3K 40 PEG 3350 20 2:1 10 mMSC (pH 5.5) 86 0 0 92 92 Dextran 3K 30 PEG 3350 30 1:1 10 mM SC (pH 5.5)92 0 0 88 90 Dextran 3K 20 PEG 3350 40 1:2 10 mM SC (pH 5.5) 82 0 0 8281 Dextran 3K 5.5 PEG 3350 55  1:10 10 mM SC (pH 5.5) 92 0 0 82 82Dextran 3K 55 PEG 3350 5.5 10:1  10 mM SC (pH 6.5) 104 0 0 81 80 Dextran3K 40 PEG 3350 20 2:1 10 mM SC (pH 6.5) 104 0 0 92 92 Dextran 3K 30 PEG3350 30 1:1 10 mM SC (pH 6.5) 82 0 0 82 102 Dextran 3K 20 PEG 3350 401:2 10 mM SC (pH 6.5) 82 0 0 57 78 Dextran 3K 5.5 PEG 3350 55  1:10 10mM SC (pH 6.5) 82 0 0 71 60 Dextran 3K 55 PEG 3350 5.5 10:1  10 mM PP(pH 5.5) 102 53 0 92 80 Dextran 3K 40 PEG 3350 20 2:1 10 mM PP (pH 5.5)59 0 0 92 102 Dextran 3K 30 PEG 3350 30 1:1 10 mM PP (pH 5.5) 82 0 0 96104 Dextran 3K 20 PEG 3350 40 1:2 10 mM PP (pH 5.5) 104 0 0 96 92Dextran 3K 5.5 PEG 3350 55  1:10 10 mM PP (pH 5.5) 101 0 0 92 80 Dextran3K 55 PEG 3350 5.5 10:1  10 mM PP (pH 6.5) 81 0 0 106 104 Dextran 3K 40PEG 3350 20 2:1 10 mM PP (pH 6.5) 102 0 0 92 82 Dextran 3K 30 PEG 335030 1:1 10 mM PP (pH 6.5) 82 0 0 82 88 Dextran 3K 20 PEG 3350 40 1:2 10mM PP (pH 6.5) 104 0 0 88 88 Dextran 3K 5.5 PEG 3350 55  1:10 10 mM PP(pH 6.5) 102 0 0 85 82 Dextran 3K 55 PEG 3350 5.5 10:1  10 mM HB (pH5.5) 104 46 0 74 92 Dextran 3K 40 PEG 3350 20 2:1 10 mM HB (pH 5.5) 1040 0 92 82 Dextran 3K 30 PEG 3350 30 1:1 10 mM HB (pH 5.5) 80 0 0 75 78Dextran 3K 20 PEG 3350 40 1:2 10 mM HB (pH 5.5) 96 0 0 65 94 Dextran 3K5.5 PEG 3350 55  1:10 10 mM HB (pH 5.5) 82 46 0 94 92 Dextran 3K 55 PEG3350 5.5 10:1  10 mM HB (pH 6.5) 68 0 0 72 78 Dextran 3K 40 PEG 3350 202:1 10 mM HB (pH 6.5) 87 0 0 90 68 Dextran 3K 30 PEG 3350 30 1:1 10 mMHB (pH 6.5) 84 0 0 82 60 Dextran 3K 20 PEG 3350 40 1:2 10 mM HB (pH 6.5)70 0 0 78 78 Dextran 3K 5.5 PEG 3350 55  1:10 10 mM HB (pH 6.5) 66 0 061 54 Dextran 3K 60 Poloxamer 188 3 20:1  Water (pH 5.6) 98 57 57 120128 Dextran 3K 55 Poloxamer 188 5.5 10:1  Water (pH 5.9) 78 0 0 114 128Dextran 3K 40 Poloxamer 188 20 2:1 Water (pH 6.5) 98 0 0 128 69 Dextran3K 60 Poloxamer 188 3 20:1  10 mM SC (pH 5.5) 100 0 0 103 105 Dextran 3K55 Poloxamer 188 5.5 10:1  10 mM SC (pH 5.5) 82 0 0 120 115 Dextran 3K40 Poloxamer 188 20 2:1 10 mM SC (pH 5.5) 85 0 0 130 130 Dextran 3K 60Poloxamer 188 3 20:1  10 mM SC (pH 6.5) 99 0 0 78 125 Dextran 3K 55Poloxamer 188 5.5 10:1  10 mM SC (pH 6.5) 85 0 0 128 130 Dextran 3K 40Poloxamer 188 20 2:1 10 mM SC (pH 6.5) 93 0 0 107 128 Dextran 3K 60Poloxamer 188 3 20:1  10 mM PP (pH 5.5) 90 57 57 67 130 Dextran 3K 55Poloxamer 188 5.5 10:1  10 mM PP (pH 5.5) 95 55 55 128 130 Dextran 3K 40Poloxamer 188 20 2:1 10 mM PP (pH 5.5) 120 0 0 115 115 Dextran 3K 60Poloxamer 188 3 20:1  10 mM PP (pH 6.5) 86 0 0 89 133 Dextran 3K 55Poloxamer 188 5.5 10:1  10 mM PP (pH 6.5) 98 0 0 120 103 Dextran 3K 40Poloxamer 188 20 2:1 10 mM PP (pH 6.5) 82 0 0 70 113 Dextran 3K 60Poloxamer 188 3 20:1  10 mM HB (pH 5.5) 103 0 0 104 110 Dextran 3K 55Poloxamer 188 5.5 10:1  10 mM HB (pH 5.5) 122 74 0 128 103 Dextran 3K 40Poloxamer 188 20 2:1 10 mM HB (pH 5.5) 125 59 0 103 122 Dextran 3K 60Poloxamer 188 3 20:1  10 mM HB (pH 6.5) 134 0 0 127 103 Dextran 3K 55Poloxamer 188 5.5 10:1  10 mM HB (pH 6.5) 115 0 0 128 110 Dextran 3K 40Poloxamer 188 20 2:1 10 mM HB (pH 6.5) 115 0 0 128 108 Dextran 40K 60Poloxamer 188 3 20:1  Water (pH 5.8) 87 0 0 76 78 Dextran 40K 55Poloxamer 188 5.5 10:1  Water (pH 6.0) 85 0 0 78 77 Dextran 40K 40Poloxamer 188 20 2:1 Water (pH 6.5) 128 0 0 75 90 Dextran 40K 60Poloxamer 188 3 20:1  10 mM SC (pH 5.5) 102 0 0 100 74 Dextran 40K 55Poloxamer 188 5.5 10:1  10 mM SC (pH 5.5) 115 0 0 83 115 Dextran 40K 40Poloxamer 188 20 2:1 10 mM SC (pH 5.5) 128 0 0 98 113 Dextran 40K 60Poloxamer 188 3 20:1  10 mM SC (pH 6.5) 100 0 0 98 98 Dextran 40K 55Poloxamer 188 5.5 10:1  10 mM SC (pH 6.5) 84 0 0 87 69 Dextran 40K 40Poloxamer 188 20 2:1 10 mM SC (pH 6.5) 100 0 0 134 98 Dextran 40K 60Poloxamer 188 3 20:1  10 mM PP (pH 5.5) 109 0 0 78 91 Dextran 40K 55Poloxamer 188 5.5 10:1  10 mM PP (pH 5.5) 99 0 0 98 100 Dextran 40K 40Poloxamer 188 20 2:1 10 mM PP (pH 5.5) 103 0 0 103 100 Dextran 40K 60Poloxamer 188 3 20:1  10 mM PP (pH 6.5) 110 0 0 83 98 Dextran 40K 55Poloxamer 188 5.5 10:1  10 mM PP (pH 6.5) 102 0 0 97 73 Dextran 40K 40Poloxamer 188 20 2:1 10 mM PP (pH 6.5) 115 0 0 94 115 Dextran 40K 60Poloxamer 188 3 20:1  10 mM HB (pH 5.5) 99 0 0 100 100 Dextran 40K 55Poloxamer 188 5.5 10:1  10 mM HB (pH 5.5) 115 62 0 91 72 Dextran 40K 40Poloxamer 188 20 2:1 10 mM HB (pH 5.5) 130 58 0 112 110 Dextran 40K 60Poloxamer 188 3 20:1  10 mM HB (pH 6.5) 110 0 0 98 98 Dextran 40K 55Poloxamer 188 5.5 10:1  10 mM HB (pH 6.5) 110 0 0 97 75 Dextran 40K 40Poloxamer 188 20 2:1 10 mM HB (pH 6.5) 128 0 0 110 130 PVP 17 55 PEG3350 5.5 10:1  Water (pH 6.5) 0 0 0 0 0 PVP 17 40 PEG 3350 20 2:1 Water(pH 4.6) 80 0 0 70 70 PVP 17 30 PEG 3350 30 1:1 Water (pH 5.0) 80 0 0 6262 PVP 17 20 PEG 3350 40 1:2 Water (pH 5.4) 68 0 0 66 66 PVP 17 5.5 PEG3350 55  1:10 Water (pH 4.1) 47 0 0 54 54 PVP 17 55 PEG 3350 5.5 10:1 10 mM SC (pH 5.5) 92 0 0 78 78 PVP 17 40 PEG 3350 20 2:1 10 mM SC (pH5.5) 76 0 0 69 69 PVP 17 30 PEG 3350 30 1:1 10 mM SC (pH 5.5) 80 0 0 6868 PVP 17 20 PEG 3350 40 1:2 10 mM SC (pH 5.5) 92 0 0 78 78 PVP 17 5.5PEG 3350 55  1:10 10 mM SC (pH 5.5) 83 0 0 64 64 PVP 17 55 PEG 3350 5.510:1  10 mM SC (pH 6.5) — — — — — PVP 17 40 PEG 3350 20 2:1 10 mM SC (pH6.5) — — — — — PVP 17 30 PEG 3350 30 1:1 10 mM SC (pH 6.5) — — — — — PVP17 20 PEG 3350 40 1:2 10 mM SC (pH 6.5) — — — — — PVP 17 5.5 PEG 3350 55 1:10 10 mM SC (pH 6.5) — — — — — PVP 17 55 PEG 3350 5.5 10:1  10 mM PP(pH 5.5) — — — — — PVP 17 40 PEG 3350 20 2:1 10 mM PP (pH 5.5) — — — — —PVP 17 30 PEG 3350 30 1:1 10 mM PP (pH 5.5) — — — — — PVP 17 20 PEG 335040 1:2 10 mM PP (pH 5.5) — — — — — PVP 17 5.5 PEG 3350 55  1:10 10 mM PP(pH 5.5) — — — — — PVP 17 55 PEG 3350 5.5 10:1  10 mM PP (pH 6.5) — — —— — PVP 17 40 PEG 3350 20 2:1 10 mM PP (pH 6.5) — — — — — PVP 17 30 PEG3350 30 1:1 10 mM PP (pH 6.5) — — — — — PVP 17 20 PEG 3350 40 1:2 10 mMPP (pH 6.5) — — — — — PVP 17 5.5 PEG 3350 55  1:10 10 mM PP (pH 6.5) — —— — — PVP 17 55 PEG 3350 5.5 10:1  10 mM HB (pH 5.5) 92 42 42 54 54 PVP17 40 PEG 3350 20 2:1 10 mM HB (pH 5.5) 92 0 0 98 98 PVP 17 30 PEG 335030 1:1 10 mM HB (pH 5.5) 109 0 0 112 112 PVP 17 20 PEG 3350 40 1:2 10 mMHB (pH 5.5) 84 0 0 61 61 PVP 17 5.5 PEG 3350 55  1:10 10 mM HB (pH 5.5)92 0 0 78 78 PVP 17 55 PEG 3350 5.5 10:1  10 mM HB (pH 6.5) 86 0 0 78 78PVP 17 40 PEG 3350 20 2:1 10 mM HB (pH 6.5) 92 0 0 102 102 PVP 17 30 PEG3350 30 1:1 10 mM HB (pH 6.5) 78 0 0 74 74 PVP 17 20 PEG 3350 40 1:2 10mM HB (pH 6.5) 104 46 46 92 92 PVP 17 5.5 PEG 3350 55  1:10 10 mM HB (pH6.5) 102 61 61 80 80 PVP 17 10 Poloxamer 188 0.25 40:1  Water (pH 4.3)64 0 0 82 78 PVP 17 5 Poloxamer 188 0.125 40:1  Water (pH 4.2) 80 0 0 7661 PVP 17 60 Poloxamer 188 3 20:1  Water (pH 4.0) 68 0 0 72 72 PVP 17 30Poloxamer 188 1.5 20:1  Water (pH 4.0) 77 0 0 68 80 PVP 17 10 Poloxamer188 0.5 20:1  Water (pH 4.3) 82 0 0 82 68 PVP 17 5 Poloxamer 188 0.2520:1  Water (pH 4.2) 79 0 0 82 62 PVP 17 55 Poloxamer 188 5 10:1  Water(pH 4.1) 78 0 0 53 71 PVP 17 27 Poloxamer 188 2.7 10:1  Water (pH 4.1)82 0 0 53 81 PVP 17 48 Poloxamer 188 12 4:1 Water (pH 4.1) 73 0 0 82 65PVP 17 24 Poloxamer 188 6 4:1 Water (pH 4.1) 78 0 0 53 65 PVP 17 10Poloxamer 188 2.5 4:1 Water (pH 4.3) 78 0 0 82 82 PVP 17 5 Poloxamer 1881.25 4:1 Water (pH 4.3) 80 0 0 68 68 PVP 17 40 Poloxamer 188 20 2:1Water (pH 4.3) 74 0 0 78 72 PVP 17 20 Poloxamer 188 10 2:1 Water (pH4.4) 71 0 0 101 97 PVP 17 10 Poloxamer 188 5 2:1 Water (pH 4.4) 79 0 083 74 PVP 17 5 Poloxamer 188 2.5 2:1 Water (pH 4.4) 63 0 0 82 70 PVP 1720 Poloxamer 188 40 1:2 Water (pH 5.7) 69 0 0 61 67 PVP 17 10 Poloxamer188 20 1:2 Water (pH 5.2) 77 0 0 91 64 PVP 17 5 Poloxamer 188 10 1:2Water (pH 5.4) 82 0 0 117 68 PVP 17 2.5 Poloxamer 188 5 1:2 Water (pH5.3) 80 0 0 70 66 PVP 17 1.25 Poloxamer 188 2.5 1:2 Water (pH 5.3) 70 —— 53 47 PVP 17 0.625 Poloxamer 188 1.25 1:2 Water (pH 5.4) 73 — — 55 78PVP 17 0.3125 Poloxamer 188 0.625 1:2 Water (pH 5.2) 64 — — 62 78 PVP 170.5 Poloxamer 188 10  1:20 Water (pH 6.4) 79 0 0 58 59 PVP 17 0.25Poloxamer 188 5  1:20 Water (pH 6.4) 82 0 0 62 0 PVP 17 55 Poloxamer 1885.5 2:1 10 mM SC (pH 5.5) 86 39 0 78 82 PVP 17 40 Poloxamer 188 20 2:110 mM SC (pH 5.5) 86 41 0 88 94 PVP 17 20 Poloxamer 188 10 2:1 10 mM SC(pH 5.5) 65 65 0 101 105 PVP 17 20 Poloxamer 188 10 2:1 10 mM SC (pH6.5) 87 0 0 97 102 PVP 17 20 Poloxamer 188 10 2:1 10 mM PP (pH 5.5) 71 00 79 79 PVP 17 20 Poloxamer 188 10 2:1 10 mM PP (pH 6.5) 65 0 0 63 65PVP 17 40 Poloxamer 188 20 2:1 10 mM NaCl (pH 4.2) 104 0 0 96 104 PVP 1720 Poloxamer 188 10 2:1 10 mM NaCl (pH 4.4) 91 0 0 93 115 PVP 17 10Poloxamer 188 20 1:2 Water (pH 5.2) 77 0 0 91 64 PVP 17 20 Poloxamer 18840 1:2 10 mM SC (pH 5.5) 92 39 0 83 96 PVP 17 10 Poloxamer 188 20 1:2 10mM SC (pH 5.5) 81 71 49 97 85 PVP 17 2.5 Poloxamer 188 5 1:2 10 mM SC(pH 5.5) 104 — — 76 73 PVP 17 1.25 Poloxamer 188 2.5 1:2 10 mM SC (pH5.5) 72 — — 92 90 PVP 17 0.625 Poloxamer 188 1.25 1:2 10 mM SC (pH 5.5)102 — — 102 88 PVP 17 0.3125 Poloxamer 188 0.625 1:2 10 mM SC (pH 5.5)84 — — 78 90 PVP 17 10 Poloxamer 188 20 1:2 10 mM SC (pH 6.5) 88 0 0 7991 PVP 17 10 Poloxamer 188 20 1:2 10 mM PP (pH 5.5) 68 0 0 73 89 PVP 1710 Poloxamer 188 20 1:2 10 mM PP (pH 6.5) 88 0 0 88 88 PVP 17 60Poloxamer 188 3 20:1  10 mM HB (pH 5.5) 79 0 0 80 80 PVP 17 55 Poloxamer188 5.5 10:1  10 mM HB (pH 5.5) 92 0 0 0 0 PVP 17 40 Poloxamer 188 202:1 10 mM HB (pH 5.5) 72 0 0 92 92 PVP 17 20 Poloxamer 188 40 1:2 10 mMHB (pH 5.5) 106 42 42 102 102 PVP 17 60 Poloxamer 188 3 20:1  10 mM HB(pH 6.5) 104 46 46 100 100 PVP 17 55 Poloxamer 188 5.5 10:1  10 mM HB(pH 6.5) 112 0 0 91 91 PVP 17 40 Poloxamer 188 20 2:1 10 mM HB (pH 6.5)91 0 0 102 102 PVP 17 20 Poloxamer 188 40 1:2 10 mM HB (pH 6.5) 100 0 00 0 PVP 17 60 Poloxamer 188 3 20:1  10 mM NaCl (pH 3.0) 58 0 0 54 54 PVP17 30 Poloxamer 188 1.5 10:1  10 mM NaCl (pH 3.0) 78 0 0 80 92 PVP 17 55Poloxamer 188 5.5 10:1  10 mM NaCl (pH 4.0) 68 0 0 92 92 PVP 17 27Poloxamer 188 2.7 10:1  10 mM NaCl (pH 4.0) 76 0 0 88 83 PVP 17 48Poloxamer 188 12 4:1 10 mM NaCl (pH 4.1) 92 — — 82 82 PVP 17 24Poloxamer 188 6 4:1 10 mM NaCl (pH 4.1) 102 0 0 78 79 PVP 17 40Poloxamer 188 20 2:1 10 mM NaCl (pH 4.3) 102 — — 55 55 PVP 17 20Poloxamer 188 10 2:1 10 mM NaCl (pH 4.3) 78 0 0 88 82 PVP 17 10Poloxamer 188 20 1:2 10 mM NaCl (pH 4.7) 115 0 0 80 81 PVP 17 2.5Poloxamer 188 5 1:2 10 mM NaCl (pH 5.2) 94 — — 101 79 PVP 17 1.25Poloxamer 188 2.5 1:2 10 mM NaCl (pH 5.2) 88 — — 100 102 PVP 17 0.625Poloxamer 188 1.25 1:2 10 mM NaCl (pH 5.2) 96 — — 98 77 PVP 17 0.3125Poloxamer 188 0.625 1:2 10 mM NaCl (pH 5.2) 85 — — 76 80 PVP 17 10Polysorbate 80 0.5 20:1  Water (pH 4.2) 82 — — 81 81 PVP 17 5Polysorbate 80 0.25 20:1  Water (pH 4.2) 84 — — 77 77 PVP 17 10Polysorbate 80 2.5 4:1 Water (pH 4.4) 90 — — 82 82 PVP 17 5 Polysorbate80 1.25 4:1 Water (pH 4.4) 90 — — 104 104 PEG 3350 50 Mannitol 5 10:1 Water 0 0 0 0 0 PEG 3350 50 Mannitol 50 1:1 Water 26 — — — — PEG 3350 5Mannitol 50  1:10 Water 30 — — — — PEG 3350 60 Poloxamer 188 3 20:1 Water (pH 7.0) 0 0 0 0 0 PEG 3350 55 Poloxamer 188 5.5 10:1  Water (pH7.0) 0 0 0 0 0 PEG 3350 50 Poloxamer 188 5 10:1  Water (pH 7.0) 0 0 0 00 PEG 3350 40 Poloxamer 188 20 2:1 Water (pH 7.0) 0 0 0 0 0 PEG 3350 50Poloxamer 188 50 1:1 Water 0 0 0 0 0 PEG 3350 5 Poloxamer 188 50  1:10Water 0 0 0 0 0 PEG 3350 60 Poloxamer 188 3 20:1  10 mM SC (pH 5.5) 10166 46 94 95 PEG 3350 55 Poloxamer 188 5.5 10:1  10 mM SC (pH 5.5) 90 590 87 94 PEG 3350 40 Poloxamer 188 20 2:1 10 mM SC (pH 5.5) 101 59 0 9899 PEG 3350 60 Poloxamer 188 3 20:1  10 mM SC (pH 6.5) 70 0 0 58 70 PEG3350 55 Poloxamer 188 5.5 10:1  10 mM SC (pH 6.5) 66 0 0 58 66 PEG 335040 Poloxamer 188 20 2:1 10 mM SC (pH 6.5) 76 0 0 69 66 PEG 3350 60Poloxamer 188 3 20:1  10 mM PP (pH 5.5) 92 0 0 87 77 PEG 3350 55Poloxamer 188 5.5 10:1  10 mM PP (pH 5.5) 98 0 0 86 101 PEG 3350 40Poloxamer 188 20 2:1 10 mM PP (pH 5.5) 83 0 0 96 78 PEG 3350 60Poloxamer 188 3 20:1  10 mM PP (pH 6.5) 0 0 0 0 0 PEG 3350 55 Poloxamer188 5.5 10:1  10 mM PP (pH 6.5) 0 0 0 0 0 PEG 3350 40 Poloxamer 188 202:1 10 mM PP (pH 6.5) 0 0 0 0 0 PEG 3350 60 Poloxamer 188 3 20:1  10 mMHB (pH 5.5) 75 78 76 91 101 PEG 3350 55 Poloxamer 188 5.5 10:1  10 mM HB(pH 5.5) 98 98 63 98 73 PEG 3350 40 Poloxamer 188 20 2:1 10 mM HB (pH5.5) 72 82 65 83 89 PEG 3350 60 Poloxamer 188 3 20:1  10 mM HB (pH 6.5)85 109 101 112 92 PEG 3350 55 Poloxamer 188 5.5 10:1  10 mM HB (pH 6.5)81 101 82 136 87 PEG 3350 40 Poloxamer 188 20 2:1 10 mM HB (pH 6.5) 65106 85 120 109 PEG 3350 50 Glycine 5 10:1  Water 0 0 0 0 0 PEG 3350 50Glycine 50 1:1 Water 0 0 0 0 0 PEG 3350 5 Glycine 50  1:10 Water 0 0 0 00 ^(a)Amount of botulinum neurotoxin serotype A complex added performulation was 150 units. Total volume of formulation was 1.0 mL.^(b)Recovery is expressed as a percentage and is calculated by dividingthe potency of the active ingredient determined after reconstitutiondivided by the potency of the active ingredient determined beforeaddition to the formulation. 3 months refers to the length of time aformulation was minimally stored at the indicated temperature. 12 monthsrefers to the length of time a formulation was minimally stored at theindicated temperature. ^(c)Ambient temperature is between about 18° C.to about 22° C. ^(d)Below freezing temperature is between about −5° C.to about −20° C.

Similar increased initial recovered potency of the Clostridial toxinactive ingredient was observed with PVP 17 in combination withPolysorbate 80 (Table 4). Clostridial toxin compositions comprisingabout 5 mg to about 10 mg of PVP 17 (about 0.5% (w/v) to about 1% (w/V))as the sole excipient resulted in about 48% to about 52% recoveredpotency of a Clostridial toxin active ingredient (Table 2). However,Clostridial toxin compositions comprising about 5 mg to about 10 mg ofPVP 17 (about 0.5% (w/v) to about 1% (w/V)) and about 0.25 mg to about2.5 mg polysorbate 80 (about 0.025% (w/v) to about 0.25% (w/v)) resultedin an initial recovered potency of about 82% to about 90% (Table 4). Theenhancement of long term stability was also observed in Clostridialtoxin compositions comprising about sucrose and polysorbate 80 (seeTable 4).

Clostridial toxin pharmaceutical compositions comprising PEG 3350 and asurfactant resulted in enhanced initial recovered potency and long-termstability of the Clostridial toxin active ingredient when formulatedwith certain buffered solutions. For example, both PEG 3350 alone andPoloxamer 188 alone resulted in no detectable recovered potency of aClostridial toxin active ingredient (Table 2). Similarly, Clostridialtoxin pharmaceutical compositions comprising PEG 3350 and Poloxamer 188in water resulted in no detectable recovered potency of a Clostridialtoxin active ingredient (Table 4). Surprisingly, however, Clostridialtoxin pharmaceutical compositions comprising PEG 3350 and Poloxamer 188in buffered formulations all resulted in effective recovered potency ofthe Clostridial toxin active ingredient, and in many cases resulted inenhanced initial recovery and long-term stability (Table 4). Forexample, Clostridial toxin pharmaceutical compositions comprising about60 mg PEG 3350 (about 6% (w/v)) in about pH 5.5 sodium citrate bufferresulted in an initial recovered potency of about 76%, whereascompositions comprising about 20 mg PEG 3350 (about 2% (w/v)) in aboutpH 5.5 sodium citrate buffer resulted in an initial recovered potency ofabout 81%. However, Clostridial toxin pharmaceutical compositionscomprising about 40 mg to about 60 mg PEG 3350 (about 4% (w/v) to about6% (w/v)) and about 3 mg to about 20 mg of Poloxamer 188 (about 0.3%(w/v) to about 2% (w/v)) in about pH 5.5 sodium citrate buffer resultedin an initial recovered potencies of about 90% to about 101%. Long termstability of the Clostridial toxin active ingredient was also enhancedin these formulations (Table 4).

Similarly, Clostridial toxin pharmaceutical compositions comprisingabout 60 mg PEG 3350 (about 6% (w/v)) in about pH 6.5 sodium citratebuffer resulted in an initial recovered potency of about 57%, whereascompositions comprising about 20 mg PEG 3350 (about 2% (w/v)) in aboutpH 6.5 sodium citrate buffer resulted in an initial recovered potency ofabout 80%. However, Clostridial toxin pharmaceutical compositionscomprising about 40 mg to about 60 mg PEG 3350 (about 4% (w/v) to about6% (w/v)) and about 3 mg to about 20 mg of Poloxamer 188 (about 0.3%(w/v) to about 2% (w/v)) in about pH 6.5 sodium citrate buffer resultedin an initial recovered potencies of about 83% to about 98%. Long termstability of the Clostridial toxin active ingredient was also enhancedin these formulations (Table 4).

Clostridial toxin pharmaceutical compositions comprising a polyol and asurfactant also resulted in recovered potency of the Clostridial toxinactive ingredient. For example, both mannitol alone and Poloxamer 188alone resulted in no detectable recovered potency of a Clostridial toxinactive ingredient (Table 2). Surprisingly Clostridial toxinpharmaceutical compositions comprising mannitol and Poloxamer 188resulted in recovered potency of the Clostridial toxin active ingredient(Table 5).

Clostridial toxin pharmaceutical compositions comprising an amino acidand a surfactant also resulted in recovered potency of the Clostridialtoxin active ingredient. For example, both glycine alone and Poloxamer188 alone resulted in no detectable recovered potency of a Clostridialtoxin active ingredient (Table 2). Surprisingly Clostridial toxinpharmaceutical compositions comprising glycine and Poloxamer 188resulted in recovered potency of about 30% to about 35% of theClostridial toxin active ingredient (Table 5).

TABLE 5 Formulations using Botulinum Neurotoxin Complex^(a) - TwoExcipients with One Being a Surfactant Recovered Potency^(b) (%)Excipient 1 Excipient 2 Ambient Below Frerezing Amount AmountTemperatrue^(c) Temperature^(d) Type (mg) Type (mg) Ratio Solution (pH)Initial 3 months 12 months 3 months 12 months Poloxamer 188 50 Mannitol5 10:1  Water 30 — — — — Poloxamer 188 50 Mannitol 50 1:1 Water 33 — — —— Poloxamer 188 5 Mannitol 50  1:10 Water 35 — — — — Poloxamer 188 50Glycine 5 10:1  Water 33 — — — — Poloxamer 188 50 Glycine 50 1:1 Water26 — — — — Poloxamer 188 5 Glycine 50  1:10 Water 0 0 0 0 0 ^(a)Amountof botulinum neurotoxin serotype A complex added per formulation was 150units. Total volume of formulation was 1.0 mL. ^(b)Recovery is expressedas a percentage and is calculated by dividing the potency of the activeingredient determined after reconstitution divided by the potency of theactive ingredient determined before addition to the formulation. 3months refers to the length of time a formulation was minimally storedat the indicated temperature. 12 months refers to the length of time aformulation was minimally stored at the indicated temperature.^(c)Ambient temperature is between about 18° C. to about 22° C.^(d)Below freezing temperature is between about −5° C. to about −20° C.

Example 3 Non-Protein Stabilized Formulations—Three Excipients

Experiments were carried out to determine the effects of formulationscomprising three different non-protein excipients on Clostridial toxinactive ingredient recovery after reconstitution. The non-proteinexcipients tested were added separately or in combination with thelisted buffers or salts (Table 6). All of the formulations werecompounded, lyophilized, reconstituted and potency assessed in the samemanner, and with the same Clostridial toxin active ingredient used ineach formulation, except that each formulation was prepared withdifferent non-protein excipients or with different amounts of thenon-protein excipients.

The tested formulations were compounded, processed, stored andreconstituted as described in Example 1. Recovered potency wasdetermined using the mouse LD₅₀ bioassay described in Example 1.Recovery is expressed as a percentage and is calculated by dividing thepotency of the Clostridial toxin active ingredient in the storedreconstitution formulation by the potency of the active Clostridialtoxin ingredient determined prior to its addition into the testsolution. The results show that a Clostridial toxin pharmaceuticalcomposition comprising a Clostridial toxin complex could be stabilizedwhen the formulation comprised three non-protein excipients (Table 6).

Clostridial toxin pharmaceutical compositions comprising a sugar, anon-protein polymer and a surfactant resulted in an effective recoveredpotency and long-term stability of the Clostridial toxin activeingredient. For example, Clostridial toxin pharmaceutical compositionscomprising about 10 mg sucrose (1% (w/v)) and about 10 mg PVP 17 (1%(w/v)) exhibited an initial recovered potency of the Clostridial toxinactive ingredient of about 77% (Table 4). Likewise, Clostridial toxinpharmaceutical compositions comprising about 10 mg sucrose (about 1%(w/v)) and about 10 mg Poloxamer 188 (about 1% (w/v)) exhibited aninitial recovered potency of the Clostridial toxin active ingredient ofabout 59% (Table 4). Similarly, Clostridial toxin pharmaceuticalcompositions comprising about 10 mg to about 20 mg of Kollodon 17 (about1% (w/v) to about 2% (w/v)) and about 10 mg to about 20 mg Poloxamer 188(about 1% (w/v) to about 2% (w/v)) exhibited an initial recoveredpotency of the Clostridial toxin active ingredient of about 71% to about82% (Table 4). However, Clostridial toxin pharmaceutical compositionscomprising about 10 mg sucrose (about 1% (w/v)), about 10 mg PVP 17(about 1% (w/v)), and about 10 mg Poloxamer 188 (about 1% (w/v)),exhibited a recovered potency of the Clostridial toxin active ingredientof about 102% (Table 6). A similar increase in initial recoveredpotency, of about 89%, was observed in Clostridial toxin pharmaceuticalcompositions comprising about 15 mg sucrose (about 1.5% (w/v)), about 30mg PVP 17 (about 3% (w/v)), and about 15 mg Poloxamer 188 (about 1.5%(w/v)) (Table 6). The addition of various buffers or sodium chloride toClostridial toxin pharmaceutical compositions comprising sucrose, PVP 17and Poloxamer 188 enhanced initial recovered potency or long-termstability of the Clostridial toxin active ingredient, depending on theamounts of each excipient added (Table 6).

Clostridial toxin pharmaceutical compositions comprising two differentsugars and a surfactant resulted in an effective recovered potency andlong-term stability of the Clostridial toxin active ingredient. Forexample, compositions comprising sucrose, lactose and Poloxamer 188resulted in initial recovered potency of about 81% to about 114% (Table6). Surprisingly, Clostridial toxin pharmaceutical compositionscomprising sucrose, lactose and Poloxamer 188 enhanced initial recoveredpotency with the addition of about pH 6.5 sodium citrate buffer. Forexample, Clostridial toxin pharmaceutical compositions comprising about20 mg sucrose (about 2% (w/v)) and about 20 mg lactose (about 2% (w/v))in about pH 6.5 sodium citrate buffer resulted in 41% initial recoveredpotency (Table 3). Likewise, Clostridial toxin pharmaceuticalcompositions comprising about 20 mg sucrose (about 2% (w/v)) and about10 mg Poloxamer 188 (about 1% (w/v)) in about pH 6.5 sodium citratebuffer resulted in 90% initial recovered potency (Table 3). Similarly,Clostridial toxin pharmaceutical compositions comprising about 20 mglactose (about 2% (w/v)) and about 10 mg Poloxamer 188 (about 1% (w/v))in about pH 6.5 sodium citrate buffer resulted in 81% initial recoveredpotency (Table 3). However, compositions comprising all three excipientsin about pH 6.5 sodium citrate buffer resulted in about 99% initialrecovered potency (Table 6).

Clostridial toxin pharmaceutical compositions comprising a sugar and twodifferent non-protein polymers resulted in enhanced recovered potencyand long-term stability of the Clostridial toxin active ingredient. Forexample, Clostridial toxin pharmaceutical compositions comprising about5 mg to about 20 mg of sucrose (about 0.5% (w/v) to about 2% (w/v)) andabout 5 mg to about 15 mg PVP 17 (about 0.5% (w/v) to about 1.5% (w/v))resulted in initial recovered potency of about 58% to about 77% (Table3). Likewise, Clostridial toxin pharmaceutical compositions comprisingabout 5 mg to about 50 mg of sucrose (about 0.5% (w/v) to about 5%(w/v)) and about 5 mg to about 50 mg PEG 3350 (about 0.5% (w/v) to about5% (w/v)) resulted in initial recovered potency of about 35% to about44% (Table 3). Similarly, Clostridial toxin pharmaceutical compositionscomprising about 30 mg to about 40 mg of PVP 17 (about 3% (w/v) to about4% (w/v)) and about 20 mg to about 30 mg PEG 3350 (about 2% (w/v) toabout 2% (w/v)) resulted in initial recovered potency of about 80%(Table 4). However, compositions comprising all three excipientsresulted in about 82% to about 102% initial recovered potency (Table 6).

Clostridial toxin pharmaceutical compositions comprising two differentnon-protein polymers and a surfactant resulted in an effective recoveredpotency and long-term stability of the Clostridial toxin activeingredient. For example, Clostridial toxin pharmaceutical compositionscomprising Dextran 3K, PEG 3350 and Poloxamer 188 resulted in initialrecovered potencies of about 81% to about 104% when in water, about 88%to about 106% when in about pH 5.5 sodium citrate buffer, about 76% toabout 96% when in about pH 6.5 sodium citrate buffer, about 87% to about96% when in about pH 6.5 potassium phosphate buffer, about 82% to about106% when in about pH 6.5 potassium phosphate buffer, about 70% to about102% when in about pH 5.5 histidine buffer, and about 65% to about 102%when in about pH 6.5 histidine buffer (Table 6). Similarly, Clostridialtoxin pharmaceutical compositions comprising PVP 17, PEG 3350 andPoloxamer 188 resulted in an effective recovered potency and long-termstability of the Clostridial toxin active ingredient (Table 6).

TABLE 6 Formulations using Botulinum Neurotoxin Complex^(a) - ThreeExcipients Recovered Potency^(b) (%) Ambient Below Frerezing Excipient 1Excipient 2 Excipient 3 Temperatrue^(c) Temperature^(d) Amount AmountAmount 3 12 3 12 Type (mg) Type (mg) Type (mg) Ratio Solution (pH)Initial months months months months Sucrose 30 PVP 17 30 Poloxamer 188 310:10:1 Water (pH 4.2) 75 — — 79 62 Sucrose 15 PVP 17 15 Poloxamer 1881.5 10:10:1 Water (pH 4.6) 82 0 0 70 70 Sucrose 27.5 PVP 17 27.5Poloxamer 188 5.5 5:5:1 Water (pH 4.2) 66 0 0 65 65 Sucrose 13.5 PVP 1713.5 Poloxamer 188 2.7 5:5:1 Water (pH 4.2) 82 0 0 76 76 Sucrose 20 PVP17 10 Poloxamer 188 5 4:2.1 Water (pH 4.5) 104 59 55 110 113 Sucrose 20PVP 17 20 Poloxamer 188 10 2:2:1 Water (pH 4.4) 102 49 0 96 103 Sucrose24 PVP 17 24 Poloxamer 188 12 2:2:1 Water (pH 4.4) 88 0 0 62 61 Sucrose12 PVP 17 12 Poloxamer 188 6 2:2:1 Water (pH 4.4) 88 0 0 65 80 Sucrose30 PVP 17 15 Poloxamer 188 15 2:1:1 Water (pH 4.3) 80 0 0 115 91 Sucrose15 PVP 17 30 Poloxamer 188 15 1:2:1 Water (pH 4.3) 89 — — 84 88 Sucrose20 PVP 17 20 Poloxamer 188 20 1:1:1 Water (pH 4.6) 81 0 0 81 85 Sucrose10 PVP 17 10 Poloxamer 188 10 1:1:1 Water (pH 4.6) 102 46 0 79 92Sucrose 12 PVP 17 24 Poloxamer 188 24 1:2:2 Water (pH 4.8) 104 0 0 82 92Sucrose 10 PVP 17 20 Poloxamer 188 30 1:2:3 Water (pH 5.0) 97 49 0 10295 Sucrose 20 PVP 17 10 Poloxamer 188 5 4:2:1 10 mM SC (pH 5.5) 83 51 4973 89 Sucrose 20 PVP 17 10 Poloxamer 188 5 4:2:1 10 mM SC (pH 6.5) 10152 41 101 103 Sucrose 20 PVP 17 10 Poloxamer 188 5 4:2:1 10 mM PP (pH5.5) 85 68 41 115 101 Sucrose 20 PVP 17 10 Poloxamer 188 5 4:2:1 10 mMPP (pH 6.5) 89 69 38 103 101 Sucrose 20 PVP 17 20 Poloxamer 188 10 2:2:110 mM SC (pH 5.5) 83 51 0 97 91 Sucrose 20 PVP 17 20 Poloxamer 188 102:2:1 10 mM SC (pH 6.5) 100 0 0 87 113 Sucrose 20 PVP 17 20 Poloxamer188 10 2:2:1 10 mM PP (pH 5.5) 93 58 41 110 99 Sucrose 20 PVP 17 20Poloxamer 188 10 2:2:1 10 mM PP (pH 6.5) 63 55 0 57 89 Sucrose 15 PVP 1730 Poloxamer 188 15 1:2:1 10 mM SC (pH 5.5) 106 58 0 103 101 Sucrose 20PVP 17 20 Poloxamer 188 20 1:1:1 10 mM SC (pH 5.5) 96 — — 95 91 Sucrose12 PVP 17 24 Poloxamer 188 24 1:2:2 10 mM SC (pH 5.5) 104 65 0 100 105Sucrose 10 PVP 17 20 Poloxamer 188 30 1:2:3 10 mM SC (pH 5.5) 108 63 0107 96 Sucrose 14 PVP 17 14 Poloxamer 188 1.4 10:10:1 10 mM NaCl (pH4.1) 92 0 0 88 75 Sucrose 27.5 PVP 17 27.5 Poloxamer 188 5.5 5:5:1 10 mMNaCl (pH 4.2) 92 0 0 75 75 Sucrose 13.75 PVP 17 13.75 Poloxamer 188 2.755:5:1 10 mM NaCl (pH 4.2) 88 0 0 78 92 Sucrose 20 PVP 17 10 Poloxamer188 5 4:2:1 10 mM NaCl (pH 4.5) 107 59 51 115 113 Sucrose 20 PVP 17 20Poloxamer 188 10 2:2:1 10 mM NaCl (pH 4.4) 103 49 0 103 117 Sucrose 24PVP 17 24 Poloxamer 188 12 2:2:1 10 mM NaCl (pH 4.3) 82 0 0 85 72Sucrose 12 PVP 17 12 Poloxamer 188 6 2:2:1 10 mM NaCl (pH 4.4) 80 0 0 7584 Sucrose 20 PVP 17 20 Poloxamer 188 20 1:1:1 10 mM NaCl (pH 4.5) 82 00 92 92 Sucrose 10 PVP 17 10 Poloxamer 188 10 1:1:1 10 mM NaCl (pH 4.6)92 50 52 83 92 Sucrose 20 Lactose 20 Poloxamer 188 10 2:2:1 Water (pH5.5) 89 84 67 102 108 Sucrose 20 Lactose 20 Poloxamer 188 10 2:2:1 10 mMSC (pH 5.5) 88 85 67 87 91 Sucrose 20 Lactose 20 Poloxamer 188 10 2:2:110 mM SC (pH 6.5) 99 65 65 77 117 Sucrose 20 Lactose 20 Poloxamer 188 102:2:1 10 mM PP (pH 5.5) 114 87 73 115 115 Sucrose 20 Lactose 20Poloxamer 188 10 2:2:1 10 mM PP (pH 6.5) 89 101 58 101 114 Sucrose 20Lactose 20 Poloxamer 188 10 2:2:1 10 mM NaCl (pH 5.4) 81 101 65 115 101Sucrose 25 Glycine 25 Poloxamer 188 5 5:5:1 Water (pH 6.1) 93 82 82 8080 Sucrose 13.75 Glycine 13.75 Poloxamer 188 2.75 5:5:1 Water (pH 6.1)92 — — 95 95 Sucrose 10 PVP 17 10 PEG 3350 10 1:1:1 Water (pH 4.9) 88 5353 72 72 Sucrose 5 PVP 17 5 PEG 3350 5 1:1:1 Water (pH 4.9) 102 61 46 8282 Sucrose 10 PVP 17 20 PEG 3350 10 1:2:1 Water (pH 4.6) 92 0 0 62 62Sucrose 5 PVP 17 10 PEG 3350 5 1:2:1 Water (pH 4.6) 96 61 0 100 80Sucrose 2.5 PVP 17 5 PEG 3350 2.5 1:2:1 Water (pH 5.0) 82 0 0 82 82Lactose 40 PEG 3550 10 Poloxamer 188 10 4:1:1 Water (pH 5.6) 91 59 0 110102 Lactose 40 PEG 3550 10 Poloxamer 188 10 4:1:1 10 mM SC (pH 5.5) 9560 64 104 95 Dextran 3K 30 PEG 3550 30 Poloxamer 188 3 10:10:1 Water (pH6.6) 82 0 0 82 92 Dextran 3K 50 PEG 3550 5 Poloxamer 188 5 5:1:1 Water(pH 6.2) 90 0 0 75 82 Dextran 3K 5 PEG 3550 50 Poloxamer 188 5 1:5:1Water (pH 6.9) 81 0 0 62 62 Dextran 3K 20 PEG 3550 20 Poloxamer 188 201:1:1 Water (pH 6.8) 104 0 0 0 0 Dextran 3K 30 PEG 3550 30 Poloxamer 1883 10:10:1 10 mM SC (pH 5.5) 102 0 0 67 104 Dextran 3K 50 PEG 3550 5Poloxamer 188 5 5:1:1 10 mM SC (pH 5.5) 92 0 0 92 92 Dextran 3K 5 PEG3550 50 Poloxamer 188 5 1:5:1 10 mM SC (pH 5.5) 88 0 0 88 80 Dextran 3K20 PEG 3550 20 Poloxamer 188 20 1:1:1 10 mM SC (pH 5.5) 106 0 0 82 92Dextran 3K 30 PEG 3550 30 Poloxamer 188 3 10:10:1 10 mM SC (pH 6.5) 79 00 88 88 Dextran 3K 50 PEG 3550 5 Poloxamer 188 5 5:1:1 10 mM SC (pH 6.5)96 0 0 88 98 Dextran 3K 5 PEG 3550 50 Poloxamer 188 5 1:5:1 10 mM SC (pH6.5) 76 0 0 92 92 Dextran 3K 20 PEG 3550 20 Poloxamer 188 20 1:1:1 10 mMSC (pH 6.5) 95 0 0 102 87 Dextran 3K 30 PEG 3550 30 Poloxamer 188 310:10:1 10 mM PP (pH 5.5) 92 0 0 104 82 Dextran 3K 50 PEG 3550 5Poloxamer 188 5 5:1:1 10 mM PP (pH 5.5) 92 0 0 96 82 Dextran 3K 5 PEG3550 50 Poloxamer 188 5 1:5:1 10 mM PP (pH 5.5) 96 0 0 82 104 Dextran 3K20 PEG 3550 20 Poloxamer 188 20 1:1:1 10 mM PP (pH 5.5) 87 0 0 92 104Dextran 3K 30 PEG 3550 30 Poloxamer 188 3 10:10:1 10 mM PP (pH 6.5) 96 00 96 88 Dextran 3K 50 PEG 3550 5 Poloxamer 188 5 5:1:1 10 mM PP (pH 6.5)100 0 0 104 102 Dextran 3K 5 PEG 3550 50 Poloxamer 188 5 1:5:1 10 mM PP(pH 6.5) 106 0 0 98 82 Dextran 3K 20 PEG 3550 20 Poloxamer 188 20 1:1:110 mM PP (pH 6.5) 82 0 0 106 104 Dextran 3K 30 PEG 3550 30 Poloxamer 1883 10:10:1 10 mM HB (pH 5.5) 70 0 0 92 0 Dextran 3K 50 PEG 3550 5Poloxamer 188 5 5:1:1 10 mM HB (pH 5.5) 90 53 0 86 102 Dextran 3K 5 PEG3550 50 Poloxamer 188 5 1:5:1 10 mM HB (pH 5.5) 102 46 0 82 76 Dextran3K 20 PEG 3550 20 Poloxamer 188 20 1:1:1 10 mM HB (pH 5.5) 75 46 0 92 68Dextran 3K 30 PEG 3550 30 Poloxamer 188 3 10:10:1 10 mM HB (pH 6.5) 87 00 102 86 Dextran 3K 50 PEG 3550 5 Poloxamer 188 5 5:1:1 10 mM HB (pH6.5) 92 0 0 84 90 Dextran 3K 5 PEG 3550 50 Poloxamer 188 5 1:5:1 10 mMHB (pH 6.5) 102 0 0 106 61 Dextran 3K 20 PEG 3550 20 Poloxamer 188 201:1:1 10 mM HB (pH 6.5) 65 0 0 96 78 PVP 17 30 PEG 3550 30 Poloxamer 1883 10:10:1 Water (pH 5.1) 66 0 0 58 58 PVP 17 50 PEG 3550 5 Poloxamer 1885 5:1:1 Water (pH 4.2) 82 0 0 70 70 PVP 17 5 PEG 3550 50 Poloxamer 188 51:5:1 Water (pH 6.6) 0 0 0 0 0 PVP 17 20 PEG 3550 20 Poloxamer 188 201:1:1 Water (pH 5.4) 78 0 0 66 66 PVP 17 30 PEG 3550 30 Poloxamer 188 310:10:1 10 mM SC (pH 5.5) 82 0 0 62 62 PVP 17 50 PEG 3550 5 Poloxamer188 5 5:1:1 10 mM SC (pH 5.5) 88 0 0 78 78 PVP 17 5 PEG 3550 50Poloxamer 188 5 1:5:1 10 mM SC (pH 5.5) 96 0 0 96 96 PVP 17 20 PEG 355020 Poloxamer 188 20 1:1:1 10 mM SC (pH 5.5) 82 0 0 100 100 PVP 17 30 PEG3550 30 Poloxamer 188 3 10:10:1 10 mM SC (pH 6.5) — — — — — PVP 17 50PEG 3550 5 Poloxamer 188 5 5:1:1 10 mM SC (pH 6.5) — — — — — PVP 17 5PEG 3550 50 Poloxamer 188 5 1:5:1 10 mM SC (pH 6.5) — — — — — PVP 17 20PEG 3550 20 Poloxamer 188 20 1:1:1 10 mM SC (pH 6.5) — — — — — PVP 17 30PEG 3550 30 Poloxamer 188 3 10:10:1 10 mM PP (pH 5.5) — — — — — PVP 1750 PEG 3550 5 Poloxamer 188 5 5:1:1 10 mM PP (pH 5.5) — — — — — PVP 17 5PEG 3550 50 Poloxamer 188 5 1:5:1 10 mM PP (pH 5.5) — — — — — PVP 17 20PEG 3550 20 Poloxamer 188 20 1:1:1 10 mM PP (pH 5.5) — — — — — PVP 17 30PEG 3550 30 Poloxamer 188 3 10:10:1 10 mM PP (pH 6.5) — — — — — PVP 1750 PEG 3550 5 Poloxamer 188 5 5:1:1 10 mM PP (pH 6.5) — — — — — PVP 17 5PEG 3550 50 Poloxamer 188 5 1:5:1 10 mM PP (pH 6.5) — — — — — PVP 17 20PEG 3550 20 Poloxamer 188 20 1:1:1 10 mM PP (pH 6.5) — — — — — PVP 17 30PEG 3550 30 Poloxamer 188 3 10:10:1 10 mM HB (pH 5.5) 78 0 0 54 54 PVP17 50 PEG 3550 5 Poloxamer 188 5 5:1:1 10 mM HB (pH 5.5) 92 0 0 88 88PVP 17 5 PEG 3550 50 Poloxamer 188 5 1:5:1 10 mM HB (pH 5.5) 106 70 7082 82 PVP 17 20 PEG 3550 20 Poloxamer 188 20 1:1:1 10 mM HB (pH 5.5) 1020 0 64 64 PVP 17 30 PEG 3550 30 Poloxamer 188 3 10:10:1 10 mM HB (pH6.5) 95 0 0 92 92 PVP 17 50 PEG 3550 5 Poloxamer 188 5 5:1:1 10 mM HB(pH 6.5) 106 50 50 96 96 PVP 17 5 PEG 3550 50 Poloxamer 188 5 1:5:1 10mM HB (pH 6.5) 104 46 46 91 91 PVP 17 20 PEG 3550 20 Poloxamer 188 201:1:1 10 mM HB (pH 6.5) 110 53 53 104 104 PVP 17 25 Glycine 25 Poloxamer188 5 5:5:1 Water (pH 5.6) 79 0 0 78 78 PVP 17 13.75 Glycine 13.75Poloxamer 188 2.75 5:5:1 Water (pH 5.6) 83 — — 62 63 ^(a)Amount ofbotulinum neurotoxin serotype A complex added per formulation was 150units. Total volume of formulation was 1.0 mL. ^(b)Recovery is expressedas a percentage and is calculated by dividing the potency of the activeingredient determined after reconstitution divided by the potency of theactive ingredient determined before addition to the formulation. 3months refers to the length of time a formulation was minimally storedat the indicated temperature. 12 months refers to the length of time aformulation was minimally stored at the indicated temperature.^(c)Ambient temperature is between about 18° C. to about 22° C.^(d)Below freezing temperature is between about −5° C. to about −20° C.

Example 4 Non-Protein Stabilized Formulations—150 kDa Clostridial Toxin

Experiments were carried out to prepare multiple formulations where theClostridial toxin active ingredient contained in the formulations was a150-kDa Clostridial toxin (Table 7). The non-protein excipients testedwere added separately or in combination with the listed buffers or salts(Table 7). All of the formulations were compounded, lyophilized,reconstituted and potency assessed in the same manner, and with the sameClostridial toxin active ingredient used in each formulation, exceptthat each formulation was prepared with different non-protein excipientsor with different amounts of the non-protein excipients.

The tested formulations were compounded, processed, stored andreconstituted as described in Example 1, except that the Clostridialtoxin active ingredient added was about 150 units of a 150 kDa BoNT/A.Recovered potency was determined using the mouse LD₅₀ bioassay describedin Example 1. Recovery is expressed as a percentage and is calculated bydividing the potency of the Clostridial toxin active ingredient in thestored reconstitution formulation by the potency of the activeClostridial toxin ingredient determined prior to its addition into thetest solution. The results show that a Clostridial toxin pharmaceuticalcomposition comprising a 150-kDa Clostridial toxin could be stabilizedwhen the formulation comprised two or more non-protein excipients (Table7).

Clostridial toxin pharmaceutical compositions comprising a sugar and asurfactant resulted in an effective initial recovered potency of theClostridial toxin active ingredient. For example, both sucrose alone andPoloxamer 188 alone resulted in no detectable recovered potency of aClostridial toxin active ingredient (Table 7). Surprisingly Clostridialtoxin pharmaceutical compositions comprising sucrose in combination withPoloxamer 188 resulted in recovered potency of the Clostridial toxinactive ingredient of about 113% (Table 7). These findings regarding 150kDa BoNT/A are similar to the synergistic recovery observed with the900-kDa BoNT/A toxin complex in Examples 1-3, where Clostridial toxinpharmaceutical compositions comprising sucrose in combination withPoloxamer 188 resulted in 99% initial recovered potency (Table 3).

Clostridial toxin pharmaceutical compositions comprising lactose and/orPoloxamer 188 yielded mixed results as those seen with the 900-KdaBoNT/A toxin complex in Examples 1-3. For example, pharmaceuticalcompositions comprising lactose as the sole excipient did not result inany detectable recovered potency of the Clostridial toxin activeingredient (150 kDa BoNT/A) (Table 7). This lack of recovery wasunexpected given the finding of recovered potency of about 35% forpharmaceutical compositions comprising lactose as the sole excipientwhen the Clostridial toxin active ingredient was the 900-kDa BoNT/Atoxin complex (Table 2). Clostridial toxin pharmaceutical compositionscomprising Poloxamer 188 as the sole excipient resulted in no detectablerecovered potency of a Clostridial toxin active ingredient (Table 7), afinding similar to those discussed in Example 1. More strikingly,Clostridial toxin pharmaceutical compositions comprising lactose andPoloxamer 188 as excipients resulted in an initial recovered potency ofabout 110% (Table 7). Thus, like the 900-kDa BoNT/A toxin complex, thereis a synergistic recovery of the 150 kDa BoNT/A in pharmaceuticalcompositions comprising lactose and Poloxamer 188.

Clostridial toxin pharmaceutical compositions comprising two non-proteinpolymers also resulted in an effective initial recovered potency of theClostridial toxin active ingredient. For example, Clostridial toxinpharmaceutical compositions comprising Dextran 40K and/or Poloxamer 188also yielded comparable results as those seen with the 900-kDa BoNT/Atoxin complex in Examples 1-3. For example, recovery of the 150 kDaBoNT/A was observed in pharmaceutical compositions comprising Dextran40K and Poloxamer 188, although the initial recovered potency was lowerfor the 150 kDa BoNT/A (compare about 50% initial recovered potency ofthe 150 kDa BoNT/A in Table 7 versus about 85% initial recovered potencyof the 900-kDa BoNT/A toxin complex in Table 4).

Clostridial toxin pharmaceutical compositions comprising PEG 3350 and/orPoloxamer 188 yielded somewhat different results as those seen with the900-kDa BoNT/A toxin complex in Examples 1-3. For example, initialrecovery potency of about 47% of the 150 kDa BoNT/A was observed inpharmaceutical compositions comprising PEG 3350 and Poloxamer 188 (Table7). This recovery was unexpected given the finding that no recoveredpotency was detected for pharmaceutical compositions comprising PEG 3350and Poloxamer 188 when the Clostridial toxin active ingredient was the900-kDa BoNT/A toxin complex (Table 4). However, Clostridial toxinpharmaceutical compositions comprising PEG 3350 and/or Poloxamer 188 inabout pH 5.5 sodium citrate buffer yielded comparable results as thoseseen with the 900-kDa BoNT/A toxin complex in Examples 1-3. For example,recovery of the 150 kDa BoNT/A was observed in pharmaceuticalcompositions comprising PEG 3350 and/or Poloxamer 188 in about pH 5.5sodium citrate buffer, although the initial recovered potency was lowerfor the 150 kDa BoNT/A (compare about 52% initial recovered potency ofthe 150 kDa BoNT/A in Table 7 versus about 90% initial recovered potencyof the 900-kDa BoNT/A toxin complex in Table 4). Similarly, recovery ofthe 150 kDa BoNT/A was observed in pharmaceutical compositionscomprising PEG 3350 and/or Poloxamer 188 in about pH 5.5 potassiumphosphate buffer, although the initial recovered potency was lower forthe 150 kDa BoNT/A (compare about 53% initial recovered potency of the150 kDa BoNT/A in Table 7 versus about 98% initial recovered potency ofthe 900-kDa BoNT/A toxin complex in Table 4).

TABLE 7 Formulations using 150 kDa Botulinum Neurotoxin^(a) Excipient 1Excipient 2 Recovered Amount Amount Potency^(b) Type (mg) Type (mg)Ratio Solution (pH) (%) Poloxamer 188 50 — — — Water (pH 6.5) 0Poloxamer 188 20 — — — Water (pH 6.5) 0 Sucrose — — Water (pH 6.0) 0Sucrose 60 Poloxamer 188 6 10:1 Water (pH 6.5) 113 Lactose 60 — — Water(pH 4.4) 0 Lactose 60 Poloxamer 188 6 10:1 Water (pH 4.7) 110 Dextran40K 60 — — — Water (pH 5.0) 0 Dextran 40K 60 Poloxamer 188 6 10:1 Water(pH 5.8) 50 Dextran 40K 60 Poloxamer 188 6 10:1 10 mM SC (pH 5.5) 46Dextran 40K 60 Poloxamer 188 6 10:1 10 mM SC (pH 7.2) 0 Dextran 40K 60Poloxamer 188 6 10:1 10 mM PP (pH 5.5) 49 Dextran 40K 60 Poloxamer 188 610:1 10 mM PP (pH 7.2) 50 PEG 3350 60 — — — Water (pH 6.6) 0 PEG 3350 60— — — 10 mM SC (pH 5.5) 0 PEG 3350 60 — — — 10 mM PP (pH 5.5) 0 PEG 335060 Poloxamer 188 6 10:1 Water (pH 6.8) 47 PEG 3350 60 Poloxamer 188 610:1 10 mM SC (pH 5.5) 52 PEG 3350 60 Poloxamer 188 6 10:1 10 mM SC (pH7.2) 0 PEG 3350 60 Poloxamer 188 6 10:1 10 mM PP (pH 5.5) 53 PEG 3350 60Poloxamer 188 6 10:1 10 mM PP (pH 7.2) 0 ^(a)Amount of 150 kDa botulinumneurotoxin serotype A added per formulation was 150 units. Total volumeof formulation was 1.0 mL. ^(b)Recovery is expressed as a percentage andis calculated by dividing the potency of the active ingredientdetermined after reconstitution divided by the potency of the activeingredient determined before addition to the formulation.

Example 5 Non-Protein Stabilized Formulations—Re-Targeted ClostridialToxin

Experiments were carried out to prepare multiple formulations where theClostridial toxin active ingredient contained in the formulations was are-targeted Clostridial toxin (Table 8). The non-protein excipientstested were added separately or in combination with the listed buffersor salts (Table 6). All of the formulations were compounded,lyophilized, reconstituted and potency assessed in the same manner, andwith the same Clostridial toxin active ingredient used in eachformulation, except that each formulation was prepared with differentnon-protein excipients or with different amounts of the non-proteinexcipients.

The tested formulations were compounded, processed, stored andreconstituted as described in Example 1, except that the Clostridialtoxin active ingredient added was about 250 ng of a 100 kDa re-targetedBoNT/A, where the modification was the substitution of the BoNT/Abinding domain with an opioid ligand, see e.g., Steward, L. E. et al.,Modified Clostridial Toxins with Enhanced Translocation Capabilities andAltered Targeting Activity For Non-Clostridial Toxin Target Cells, U.S.patent application Ser. No. 11/776,075 (Jul. 11, 2007); Dolly, J. O. etal., Activatable Clostridial Toxins, U.S. patent application Ser. No.11/829,475 (Jul. 27, 2007); Foster, K. A. et al., Fusion Proteins,International Patent Publication WO 2006/059093 (Jun. 8, 2006); andFoster, K. A. et al., Non-Cytotoxic Protein Conjugates, InternationalPatent Publication WO 2006/059105 (Jun. 8, 2006), each of which isincorporated by reference in its entirety.

To determine the recovered potency of a retargeted Clostridial toxin,the reconstituted formulation was assayed by a in vitro light chainassay. In this assay, the solid formulation is reconstituted in 1.0 mLof digestion buffer comprising 2 mM DTT, 300 μM ZnCl₂, and 50 mM HEPES(pH 7.4) and incubated at 37° C. for 30 minutes. After the incubation,500 μL the incubated formulation is transferred to a new tube and 5.0 μLof 200 μM of a quench-release fluorescent substrate (SNAPTIDE® 520) wasadded. This mixture is incubated at 30° C. for about 18 to about 20hours to allow for the Clostridial toxin active ingredient to digest thequench-release fluorescent substrate. The reaction is stopped by adding25 μL of 5% TFA to the digestion mixture. The quenched digestion mixturewas then analyzed by routine reversed-phase high performance liquidchromatography (RP-HPLC) methods to separate and measure the amount ofquench-release fluorescent substrate cleaved by the reconstitutedformulation. For this RP-HPLC analysis, the quenched digestion mixturewas transferred to HPLC vials and 25 μL of this mixture was injectedinto the column (Waters SYMMETRY 300™ C18, 3.5 μm, 4.6×150 mm) set at aflow rate of 1.0 mL/min and a column temperature of 35° C. The run timewas 20 minutes with a 5 minute injection delay. The gradient mobilephase was Solution A, comprising 0.1% TFA in water, and Solution B,comprising 0.1% TFA in acetonitrile. The gradient program was asfollows: 0-10 minutes 90% A and 10% B, 10-15 minutes 80% A and 20% B,and 15-20 minutes 100% B. The multi-wavelength fluorescent detector wasset to an excitation wavelength of 322 nm and an emission wavelength of420 nm and data was collected and analyzed using standard software.Cleavage products were identified by retention time using fluorescentdetections and quantitated by peak area. Cleaved quench-releasefluorescent substrate typically eluted at a retention time of 5.7minutes.

Recovery is expressed as a percentage and is calculated by dividing thepotency of the Clostridial toxin active ingredient in the storedreconstitution formulation by the potency of the active Clostridialtoxin ingredient determined prior to its addition into the testsolution. Clostridial toxin pharmaceutical composition comprising are-targeted Clostridial toxin could be stabilized when the formulationcomprised two or more non-protein excipients in a manner similar to the900-kDa BoNT/A toxin complex and the 150 kDa BoNT/A.

The results showed that a Clostridial toxin pharmaceutical compositionscomprising a sugar and a surfactant resulted in an effective initialrecovered potency of the Clostridial toxin active ingredient. Forexample, Clostridial toxin pharmaceutical compositions comprisingsucrose or lactose in combination with Poloxamer 188 resulted inrecovered potency of the re-targeted Clostridial toxin similar to theresults observed with the 900-kDa BoNT/A toxin complex (see Examples1-3) and the 150 kDa BoNT/A (Example 4).

The results also showed that a Clostridial toxin pharmaceuticalcompositions comprising a non-protein polymer and a surfactant alsoresulted in an effective initial recovered potency of the Clostridialtoxin active ingredient. For example, Clostridial toxin pharmaceuticalcompositions comprising Dextran 40K or PEG 3550 in combination withPoloxamer 188 resulted in recovered potency of the re-targetedClostridial toxin similar to the results observed with the 900-kDaBoNT/A toxin complex (see Examples 1-3) and the 150 kDa BoNT/A (Example4).

What is claimed:
 1. A method to stabilize a Clostridial neurotoxin,comprising: providing an aqueous solution comprising a Clostridialneurotoxin and a non-mammalian protein excipient; and compounding thesolution with one or more additional non-protein excipients to provide aformulation comprising the neurotoxin in stable form.
 2. The method ofclaim 1, wherein compounding comprises adding the one or more additionalnon-protein excipients to water to form a solution and adding theaqueous solution thereto.